2-azapurine compounds and their use

ABSTRACT

Within oligonucleotides, 2-azapurine and especially 2-azaadenine bases form specifically base pairs with guanine. This base pair is of analogous stability as an adenine-thymine but less stable than a guanine-cytosine base pair. Therefore, the incorporation of 2-azaadenine residues into oligonucleotides instead of cytosine leads specifically to hybridization complexes with nucleic acids with homogenous stability. This is useful for the adaptation of the stabilities of different oligonucleotide sequences in all kinds of hybridization techniques, for example in oligomer chip technology.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is divisional application of application Ser. No.11/441,755, filed on May 25, 2006, which is a continuation of U.S.application Ser. No. 10/070,340, filed Jun. 4, 2002 (now U.S. Pat. No.7,169,553); which is a national stage application filed under 35 USC§371 of PCT/EP00/08371, filed Aug. 28, 2000; which claims benefit andpriority of European Patent Office application number 99116767.7, filedAug. 30, 1999.

The present invention is directed to a nucleic acid binding compoundcomprising 2-azapurines, a compound useful for the preparation of suchcompound, a binding product of this nucleic acid binding compound with anucleic acid, a method for the determination of a nucleic acid usingsaid compound, and several uses of 2-azapurine compounds.

Some 2-azapurine compounds are known in the art. In Chemistry ofNucleosides and Nucleotides (Vol. 2, p. 288-297, and p. 319) there areshown some examples of 2-azapurine nucleosides. However, there is nodisclosure on 7-deaza-2-azapurine nucleosides.

In Biochimica et Biophysica Acta, 520 (1978), p. 441-451, there isdisclosed the enzymatic synthesis of 2-azaadenosine-5′-diphosphate and2-aza inosine-5′-diphosphate and their polymerization to homopolymersusing E. coli polynucleotide phosphorylase. This method is complex andit is not capable of producing mixed sequences. Furthermore, there wasreported on the capability of the homopolymers to form double- andtriple-stranded complexes with other homopolymers. Those compounds onlycontaining 2-azaadenosine as base have proved to be UV sensitive andlabile.

In J. Org. Chem. 1995, 60, 6262-6269, there is disclosed the synthesisand biophysical and biological properties of oligonucleotides containing2-aza-2′-deoxyinosine. There is disclosure on a 2-aza inosinenucleoside, which is modified by a particular photochemically cleavableprotecting group at one of the ring nitrogen atoms. However, as can beseen from table 1 on page 6266, I^(Az) does not differentiate wellbetween the natural nucleobases. As can be seen further, the stabilityof the base pair I^(Az)/G is 13° less than the regular base pair C/G and9° less than the base pair A/T. The replacement of C by I^(Az) wouldtherefore not be suitable for mimicking the stability of a A/T basepair. In Liebigs Ann. Chem. 1990, 647-651, there are disclosed2-azaadenine and a nucleoside of methylthioimidazotriazine as well asthe corresponding methoxy compound. No phosphates or phosphoramiditesare disclosed. Furthermore, there is no disclosure on how to prepareoligonucleotides containing 2-aza-adenosine at specific positions. Theirhybridization behaviour is not disclosed. Furthermore, there is nodisclosure on 2-aza-2′-deoxyadenosinetriphosphate.

The present invention is particularly useful in nucleic aciddeterminations, for example in analytics, especially in the field ofhealth care. Nucleic acids have been found to be useful analytes for thedetermination of the presence or absence of genes or micro-organisms inhuperson body fluids, food or environment. Nucleic acid analysis hasfound widespread use after the introduction of nucleic acidamplification, like the Polymerase Chain Reaction (PCR, see U.S. Pat.No. 4,683,202). Thus, sufficient amounts of nucleic acids are availablefrom each sample. The nucleic acids can be determined from thispretreated sample using a variety of different techniques, dependentfrom the particular purpose. Most assays require the use of a probewhich is either immobilized or immobilizable or is labelled byattachment of one or more reporter groups. A reporter group has thecharacteristics to be itself capable to be determined or it can bereacted with reagents that make the probe determinable via said reportergroup. Thus, for example, probes that are labelled by reporter groupscan be determined, as can be hybrids that contain the probe and anucleic acid to be determined. In case of immobilized probes, the hybridbetween the probe and the nucleic acid to be determined is determined atthe solid phase to which the probe is bound. In a particular form ofassays, not only one nucleic acid having a specific sequence, but alarge number of nucleic acids of different sequence is determined. Forthis purpose, the probes are immobilized in tiny spots in an array on aflat surface such as a glass chip (EP-A-0 476 014 and TIBTECH (1997),Vol. 15, 465-469).

The basic principle of using oligonucleotide arrays was first proposedin the late 1980s when the concept of determining a DNA sequence byhybridization to a comprehensive set of oligonucleotides (SBH,sequencing by hybridization) was developed.

There are persony proposals to include modified or non-naturalheterocyclic groups instead of the natural nucleobases. Examples of suchnon-natural groups are 7-deaza-dGTP which, when introduced into anucleic acid replacing dGTP reduces band compressing in sequencing gels(EP-B-0 286 028).

Nucleic acid determinations generally suffer from the problem that thebase pairing possibilities between the natural bases A and T and C and Ghave different stability. This can be attributed to the differentcapability of the bases to form hydrogen bonding. Thus, the dA-dT-basepair has two hydrogen bridges, while the dG-dC-base pair has 3 hydrogenbridges. This results in different melting temperatures (Tm) of hybrids,depending on the GC content. The higher the GC content, the higher theTm. In routine nucleic acid analysis, however, there would be the wishto equalize the Tm for nucleic acids of the same length, or evenindependent from the length of the nucleic acid or the binding region inorder to be in the position to apply similar hybridization conditionsfor all assays. This is particularly necessary for assays using arrays,as on such arrays the hybridizing conditions for each probe must beidentical.

One solution was the use of low hybridization temperatures. Under suchconditions, persony nucleic acids having a low degree of base sequencecomplementarity will bind to the probe. This is called unspecificbinding which does not allow discrimination between similar sequences.

Another proposal was directed to the use of chemical reagents in thehybridization mixture, for example the addition of tetramethylammoniumchloride (TMAC). This reagent reduces the difference between thestability of dG-dC and dA-dT basepairs but the effect is insufficientfor short oligonucleotides. Further the addition of salts such as TMACmay not be wellcome as it complicates optimization of the assay.

Another proposal was directed to the use of different concentrations ofeach different (immobilized) probe in one assay. This was found to betechnically complex if not impossible on a chip surface.

As a further option the substitution of ribonucleotides in anoligonucleotide composed of deoxyribonucleotides, and vice versa wasapplied for the adaptation of DNA stability, Hoheisel (1996), NucleicAcids Res. 24, 430-432.

All proposals known now have some disadvantages. Therefore, there isstill a need to provide probes the Tm of which is not very dependentfrom their GC content.

FIG. 1 shows how z²A_(d) is capable of base pairing with a dG in anotherstrand of a nucleic acid.

In FIG. 2 there are shown different compounds of the invention, like2-aza-2′-deoxyadenosine (2), the corresponding triphosphate (11), aphosphoramidite for the introduction of 2-aza-2′-deoxyadenosine intooligonucleotides during conventional chemical automated synthesis (10 b)and a H-phosphonate (12).

Is FIG. 3 shows compounds useful in the present invention. The purinenumbering for compound 2 and the systematic numbering for compound 3 isgiven.

FIG. 4 shows a route for synthesis of 2-aza-2′-deoxy-adenosine.

FIG. 5 shows compounds of the invention.

FIG. 6 shows a comparison of the binding of z²A_(d) with dG and dG withisoG_(d) in antiparallel binding.

FIG. 7 shows that z²A_(d) can also bind in a parallel mode to isoG_(d),can bind to dC in parallel mode when protonated, and to iSOC_(d) inantiparallel mode if protonated.

The subject of the present invention is a nucleic acid binding compoundcomprising a backbone, said backbone having attached heterocyclic groupscapable of base pairing to natural nucleobases at least one of saidheterocyclic groups being one of the naturally occurring nucleobasescharacterized in that at least one other of said heterocyclic groups isa group of the general formula I

-   -   wherein    -   W is selected independently from X, Y and Z from the group        consisting of N and CR²,    -   Z is selected from the group consisting of N and C with the        proviso that        -   if Z is N, then            -   X independently from W and Y is selected from the group                consisting of N and CR³, and            -   Y independently from W and X is selected from the group                consisting of N and CR⁴,            -   and the bond between X and Y is a double bond and the                bond between Y and Z is a single bond, and            -   if Z is C, then            -   X is NR³³, and            -   Y is selected from the group consisting of N and CR⁴ and                the bond between Z and Y is a double bond and the bond                between X and Y is a single bond,    -   R¹, R², R³ and R⁴ are independently selected from the group        consisting of —H, -halogen, —OR¹³, —SR¹⁹, —(C₁-C₁₀)-alkyl,        —(C₂-C₁₀)-alkenyl, —(C₂-C₁₀)-allynyl, —NO₂, —NR⁵R⁶, -cyano, and        —C(═O)R¹¹,    -   R¹¹ is selected from the group consisting of —OH,        —(C₁-C₆)-alkoxy, —(C₆-C₂₂)-aryloxy, and NHR¹²,    -   R⁵, R⁶, R¹², R¹³, R¹⁹ and R³³ are selected independently from        the group consisting of —H, —(C₁-C₁₀)-alkyl, —(C₂-C₁₀)-alkenyl,        —(C₂-C₁₀)-alkinyl, —(C₆-C₂₂)-aryl, a protecting group and a        reporter group,    -   r and s are independently of each other an integer of 1 to 18,    -   D is the position of attachment of the group to the rest of the        nucleic acid binding compound, and    -   said alkyl, alkenyl and alkynyl being unsubstituted or        substituted by one or more moieties selected from the group        consisting of -halogen, —SH, —S—(C₁-C₆)-alkyl, —(C₁-C₆)-alkoxy,        —OH, —NR⁵R⁶, —COR¹¹, —NH—CONR⁵R⁶, —NH—CSNR⁵R⁶ and        —[O—(CH₂)_(r)]_(s)—NR⁵R⁶.

By way of example, in the following there is given an explanation of theadvantageous property of the compounds of the invention by showing themat the example of 2-aza-2′-deoxyadenosine (2, z²A_(d)) which formsspecifically stable base pairs with 2′-deoxyguanosine (dG) but much lessstable base pairs with 2′-deoxythymidine (dT), 2′-deoxycytidine (dC) and2′-deoxyadenosine (dA). The new base pair (z²A_(d)-dG) is of analogousstability as a regular dA-dT base pair.

In order to equalize Tm, in a nucleic acid binding compound one or moreC in a strand complementary to a G in the nucleic acid to be determinedcould be replaced by a z²A_(d). The oligonucleotide would then bindspecifically to the target sequence containing dG opposite to z²A_(d)but with the stability of a dA-dT and not a dG-dC base pair. Thisgeneral principle of course is not limited to z²A_(d), as bases showingthe same characteristics in the 6-membered ring would be expected tohave the same properties based on the above explanation due to theircontaining the 2-azapurine structure. Particularly, the farer the partof the heterocyclic group from the part participating in the basepairing, the more tolerant will the oligomer be over modifications inthe chemical structure, for example the attachment of groups to thispart of the heterocyclic rings. In the following, when reference is madeto the modified base (of the invention), there is made reference to aheterocyclic group according to general formula I.

The broken line in formula I indicates that there are severalpossibilities, depending upon the definitions of X, Y and Z, to localizea double bond. It is apparent to the person skilled in art that thechoice of a specific definition of Z will require a double bond to beeither between Z and Y or between X and Y. It is further evident thatthere will be not each a double bond between X and Y and Y and Z.

Halogen means a fluoro, chloro, bromo or iodo group. The most preferredhalogen groups are —Cl and —Br.

Alkyl groups are preferably chosen from alkyl groups containing from 1to 10 carbon atoms, either arranged in linear, branched or cyclic form.The actual length of the alkyl group will depend on the steric situationat the specific position where the alkyl group is located. If there aresteric constraints, the alkyl group will generally be smaller, themethyl and ethyl group being most preferred. All alkyl, alkenyl andalkynyl groups can be either unsubstituted or substituted. Substitutionby hetero atoms as outlined above, will help to increase solubility inaqueous solutions.

Alkenyl groups are preferably selected from alkenyl groups containingfrom 2 to 10 carbon atoms. For the selections similar considerationsapply as for alkyl groups. They also can be linear, branched and cyclic.The most preferred alkenyl group is the ethylene group.

Alkynyl groups have preferably from 2 to 10 carbon atoms. Again, thosecarbon atoms can be arranged in linear, branched and cyclic personner.Further, there can be more than one triple bond in the alkynyl group.The most preferred alkynyl group is the 3-propargyl-group.

Alkoxy groups preferably contain from 1 to 6 carbon atoms and areattached to the rest of the moiety via the oxygen atom. For the alkylgroup contained in the alkoxy groups, the same considerations apply asfor alkyl groups. The most preferred alkoxy group is the methoxy group.

Aryloxy groups preferably contain from 6 to 20 carbon atoms. Thosecarbon atoms may be contained in one or more aromatic rings and furtherin side chains (for example, alkyl chains) attached to the aromaticmoiety. Preferred aryloxy group are the phenoxy and the benzoxy group.

Preferred O-protecting groups in R¹⁴ are the aroyl groups, the acylgroups and the silyl groups. Among these most preferred is the benzoylgroup.

Preferred silyl groups are the trialkylsilyl groups, like triethylsilyl.

Any atom in the definitions within the formulae presented herein is notlimited to a specific isotope. Thus, a phosphorous atom (P) can eithermean the regular ³¹P or the radioactive ³²P or a mixture thereof. Thesame applies for hydrogen (H/D/T), carbon (C), iodine (Cl, Br, I) andnitrogen (N).

Preferred group —NR⁵R⁶ in the definition of R², R³ and R⁴ is the —NH₂group. In this case, it is evident that during chemical synthesis ofcompounds containing such group of formula I one of the hydrogen atomsof this amino group might be protected by suitable amino protectinggroup. Such protecting groups are generally known to a person skilled inthe art.

The same applies for the definitions of R¹.

During chemical synthesis, any groups —OH, —SH and —NH₂ (including thosegroups in reporter groups) should be protected by suitable protectinggroups. Further, during chemical synthesis, the compound will beattached for convenience to a solid phase. In these cases, thedefinitions of the substituents given above will be selectedaccordingly.

A protecting group is a chemical group that is attached to a functionalmoiety (for example to the oxygen in a hydroxyl group, replacing thehydrogen) to protect the functional group from reacting in an undesiredway. A protecting group is further defined by the fact that it can beremoved without destroying the biological activity of the moleculeformed, here the binding of the nucleic acid binding compound to anucleic acid. Suitable protecting groups are known to a person skilledin the art. Especially preferred protecting groups for example forhydroxyl groups at the 5′-end of a nucleotide or oligonucleotide areselected from the trityl group, for example dimethoxytrityl.

Preferred protecting groups at exocyclic amino groups in formula I arethe acyl groups, most preferred the benzoyl group (Bz), phenoxyacetyl oracetyl or formyl, and the N,N-dialkylformamidine group, preferentiallythe dimethyl-, diisobutyl-, and the di-n-butylformamidine group.

The nucleic acid binding compound according to the invention preferablyhas a length of less than 100 subunits, more preferably of from 10 to 30subunits. In order to be active as nucleic acid binding compound, thesubstituents should be chosen such that hydrogen bonds to heterocyclicgroups at the nucleic acid to be bound are enabled, preferably by WatsonCrick base pairing and/or in the way as disclosed in FIG. 1. Compoundsin which the substituents do not enable such preferred hydrogen bonding,can be useful as intermediates for the preparation of nucleic acidbinding compounds. Preferred nucleic acid binding compounds of theinvention are those which are chemically synthesized.

If the nucleic acid binding compound is to be used as a probe for thedetermination of a nucleic acid, or any other identification of thecompound or the nucleic acid is intended, any of the substituents areselected such as to contain a reporter group. While as persony reportergroups can be attached as useful to label the nucleic acid compoundsufficiently, it is preferred to attach only a limited number ofreporter groups to a single subunit, such that recognition of nucleicacids, affinities to nucleic acids and solubility is not affected suchthat the probe would not be useful in hybridization assays. In a verypreferred case, there will be only from 1 to 4, most preferably 1 or 2or most preferred only one reporter group in each nucleic acid bindingcompound. There are formats for the nucleic acid determination whichrequire more than one reporter group attached to the probe. An examplefor such formats is disclosed in WO92/02638. In this case, one of thereporter groups will be a fluorescence emitter, while the other is afluorescence quencher.

Reporter groups are generally groups that make the nucleic acid bindingcompound as well as any nucleic acids bound thereo distinguishable fromthe remainder of the liquid, i.e. the sample (nucleic acid bindingcompounds having attached a reporter group can also be termed labellednucleic acid binding compounds, labelled probes or just probes). Thisdistinction can be either effected by selecting the reporter group fromthe group of directly or indirectly detectable groups or from the groupsof immobilized or immobilizable groups. Directly detectable groups arefor example fluorescent compounds, like fluorescein and its derivatives,like hexachlorofluorescein and hexafluorofluorescein, rhodamines,psoralenes squaraines, porphyrines, fluorescent particles,bioluminescent compounds, like acridinium esters and luminol, or thecyanine dyes, like Cy-5. Examples of such compounds are disclosed in EP0 680 969. Further, spin labels like TEMPO, electrochemically detectablygroups, ferrocene, viologene, heavy metal chelates andelectrochemiluminescent labels, like ruthenium bispyridyl complexes, andnaphthoquinones, quencherdyes, like dabcyl, and nuclease activecomplexes, for example of Fe and Cu, are useful detectable groups.Indirectly detectable groups are groups that can be recognized byanother moiety which is directly or indirectly labelled. Examples ofsuch indirect detectable groups are for example haptens, likedigoxigenin or biotin. Digoxigenin for example can be recognized byantibodies against digoxigenin. Those anibodies may either be labelleddirectly or can be recognized by labelled antibodies directed againstthe (antidigoxigenin) antibodies. Formats based on the recognition ofdigoxigenin are disclosed in EP-B-0 324 474. Biotin can be recognized byavidin and similar compounds, like streptavidin and other biotin bindingcompounds. Again, those compounds can be labelled directly orindirectly.

The reporter group can further be a nucleotide sequence which does notinterfere with other nucleotide sequences in the sample. The sequencecan therefore be specifically recognized by nucleotide containing acomplementary sequence. This nucleotide sequence can be labelleddirectly or indirectly or can be immobilizable or immobilized.

A reporter group can further be a solid phase. Attachment of the nucleicacid binding compound with solid phase can be either directly orindirectly as pointed out above for the detectable group.

Direct labelling can be effected by covalent coupling of a nucleic acidbinding compound to a reactive group on the solid phase, i.e. preferablyvia a linker. Indirect labelling can be made similar as disclosed abovefor the detectable groups. Preferably, indirect attachment isnon-covalently by biospecific interactions, for example those selectedfrom the group of hapten-antibody, vitamin-receptor and nucleicacid-complementary nucleic acid. Again, those interactions and their usein nucleic acid assays is known to a person skilled in the art.

Solid phases that are useful for immobilization of the probe accordingto the invention are preferably selected from the group of polystyrene,polyethylene, polypropylene, glass and TiO₂. The formats of such solidphases can be selected according to the needs of the instrumentation andformat of the assay. For example, a solid phase may assume the form of abead or a vessel.

The term reporter group and the specific embodiments preferably includea linker which is used to connect the moiety intended to be used (theactual solid phase or the fluorophoric moiety, to the position ofattachment as the reporter group. The linker will provide flexibilitysuch that the nucleic acid binding compound can bind the nucleic acidsequence to be determined without major hindrance by the solid phase.Linkers, especially those that are not hydrophobic, for example based onconsecutive ethylenoxy units, for example as disclosed in DE 3943522 areknown to a person skilled in the art.

From the above explanation, it becomes clear that the invention wouldstill work, even if the backbone of the probe is not an oligonucleotidein the strict sense. There were described in the last years nucleicbinding compounds that have similar properties like oligonucleotides,but differ in their backbone. The backbone is generally considered to bethe part of the nucleic acid binding compound that bears the bases,mostly in linear personner, bound to identical or not identicalsubunits. The most popular backbone is the naturally occurring sugarphosphate backbone of nucleic acids (containing either ribonucleosidesubunits (RNA), deoxyribonucleoside subunits (DNA) or peptide nucleicacid subunits (PNA)). Therefore, in a preferred embodiment, the backbonecomprises sugar and phosphate moieties. In a further preferredembodiment, the sugar configuration is selected from the groupconsisting of the α-D-, β-D-, α-L- and β-L- configurations, mostpreferred the compound contains at least one2′-deoxy-β-D-erythro-pentofuranosyl moiety or one β-D-ribofuranosylmoiety.

Preferred, D is the glycosid C-1 of a sugar moiety of the compoundaccording to the invention. Preferred compounds of formula VI are thosewherein R¹ is NH₂, W is N, Z is N, Y is C, X is N and R¹⁴ is H.

Further preferred nucleic acid binding compounds contain at least onegroup of formula I, wherein R¹ is the group —NR²⁰R²¹, which are either2-aza-adenosine or derivatives thereof As derivatives of 2-aza-adeninethere are considered here compounds that provide hydrogen bonding viathe same atoms as 2-aza-adenine to G and dG in a nucleic acid bound tothe nucleic acid binding compound of the invention. The most preferredgroup of formula I is 2-aza-adenine, bound to the backbone via the N⁹atom. Those groups both discriminate clearly between the naturalnucleobases and in addition provide a very similar stability as the A-Tbase pair.

The nucleic acid binding compound will be constructed such that itcontains a nucleobase sequence which is substantially complementary tothe nucleic acid to be determined or the nucleic acid to which it isintended to be bound by base pairing. As those nucleic acids willusually contain at least once any of the naturally occurring nucleobasesAde, Cyt, Gua and Thy or Ura, the nucleic acid binding compoundaccording to the invention will also contain any of those four bases.However, according to the invention, at least one of the heterocyclicgroups in a position of the nucleic acid binding compound locatedvis-a-vis the G in the nucleic acid to be determined as replaced by theheterocyclic base of formula I. If there is more than one G in thesequence to which the nucleic acid binding compound is intended to behybridized on the nucleic acid, preferably as persony C's in the nucleicacid binding compound are chosen to be heterocyclic groups of formula Ias necessary to provide the Tm as intended.

However, the nucleic acid binding compounds of the invention display thesame base pairing selectivity also versus other heterocyclic groups inthe position located vis-à-vis the position of the nucleic acid bindingcompound at which the group of formula I is located, especially versusderivatives of G, for example c⁷G_(d), z⁸c⁷G_(d) and z⁸G_(d). In thesenominations, c⁷ means that in the 7-position there will be a carbon atomand z⁸ will correspondingly mean that in the 8-position there will be anitrogen atom. Further, derivatives of G that can be recognizedaccording to the invention, are nucleobases G which are labelled by theattachment of detectable groups, and iso G_(d).

The nucleic acid can also contain a heterocyclic group of formula Iitself. The corresponding base on the nucleic acid binding compound willpreferably be selected such as to base pair with this group, for exampleto be dG. The nucleic acid can contain natural and/or non-natural bases,for example 7-deaza-dGTP. Thus, the term nucleic acid will be construedin the present invention very broadly. Nucleic acids having mixed basesequences being preferred.

The nucleic acid binding compound according to the invention will bindto nucleic acids preferably in the antiparallel mode. However, bycarefully selecting the nucleobases of a nucleic acid and/or of thenucleic binding compound, the binding can also be forced to be in theparallel mode. Parallel hybridization of nucleic acids containing iso-Cand iso-G are for example disclosed in EP 0 624 161.

Preferred nucleic acid binding compounds are those, wherein the backbonecomprises one or more moieties of the general formula II

-   -   wherein    -   A is selected from the group consisting of O, S and        N—(C₁-C₁₀)-alkyl,    -   L is selected from the group consisting of oxy, sulfanediyl and        —NR²²—,    -   T is selected from the group consisting of oxo, thioxo and        selenoxo,    -   U is selected from the group consisting of —OH, —O-reporter        group, —SH, —S reporter group —SeH, —(C₁-C₁₀)-alkoxy,        (C₁-C₁₀)-alkyl, —(C₆-C₂₂)-aryl, —(C₆-C₁₄)-aryl-(C₁-C₁₀)-alkyl,        —NR²³R²⁴, and —O—(C₁-C₁₀)-alkyl-O—(C₁-C₁₀)-alkyl-R²⁵, or wherein        —NR²³R²⁴ can together with N be a 5-6-membered heterocyclic        ring,    -   V is selected from the group consisting of oxy, sulfanediyl or        —NR²²—,    -   R¹⁴ is selected from the group consisting of —H, —OH,        —(C₁-C₁₀)-alkoxy, —(C₂-C₁₀)-alkenyloxy, -halogen, -azido,        —O-allyl, —O-alkinyl, and —NH₂,    -   R²² is independently selected from the group of —H and        —(C₁-C₁₀)-alkyl,    -   R²³ and R²⁴ are independently selected from the group consisting        of —(C₁-C₁₀)-alkyl, —(C₁-C₂₀)-aryl,        —(C₆-C₁₄)-aryl-(C₁-C₁₀)-alkyl,        —(C₁-C₆)-alkyl-[NH(CH₂)_(c)]_(d)—NR²⁶R²⁷ and a reporter group,    -   R²⁵ is selected from the group consisting of —H, —OH, -halogen,        -amino, —(C₁-C₁₈)-alkylamino, —COOH, —CONH₂ and        —COO(C₁-C₄)-alkyl and a reporter group,    -   R²⁶ and R²⁷ are independently selected from the group consisting        from —H, —(C₁-C₆)-alkyl, and —(C₁-C₄)-alkoxy-(C₁-C₆)-alkyl and a        reporter group,    -   c is an integer from 2 to 6,    -   d is an integer from 0 to 6, and    -   B is a moiety of formula I,    -   wherein any alkyl, alkenyl and alkynyl can be substituted or        unsubstituted,    -   and any salts thereof.

The preferred definitions of the groups as defined under formula I applyto formula II and the following formulae, if not indicated otherwise.

A preferred subject of the invention is therefore a nucleic acid bindingcompound as outlined above, wherein the backbone comprises one or moremoieties of the general formula III

-   -   wherein    -   A is selected from the group consisting of O, S and        N—(C₁-C₆)-alkyl,    -   M is selected from the group consisting of oxy, sulfanediyl,        —NR²²—, —(C₁-C₁₀)-alkyl-, or —O—(C₁-C₁₀)-alkyl-O—, and        —S—(C₁-C₁₀)-alkyl-O— and —NR²²—(C₁-C₆)-alkyl-O—,    -   R²² is selected from the group of —H, —(C₁-C₁₀)-alkyl, a        protecting group and a reporter group,    -   R¹⁴ is selected from the group consisting of —H, —OH,        —(C₁-C₁₀)-alkoxy, —(C₂-C₁₀)-alkenyloxy, —(C₂-C₁₀)-alkynyloxy,        -halogen, -azido, SH, —(C₁-C₁₀)-alkylmercapto and —NH₂,    -   R¹⁵ is selected from the group consisting of —H, —(C₁-C₆)-alkyl,        —(C₂-C₁₀)-alkenyl, —(C₂-C₁₀)-alkynyl, —(C₂-C₁₀)-alkyl-carbonyl,        —(C₃-C₁₉)-alkenyl-carbonyl, —(C₃-C₁₉)-alkynyl-carbonyl,        —(C₆-C₁₄)-aryl-(C₁-C₁₀)-alkyl, a solid phase and a group of        formula IV

-   -   wherein    -   T is selected from the group consisting of oxo, thioxo and        selenoxo, and    -   U is selected from the group consisting of —OH, —O-reporter        group, —SH, —SeH, —(C₁-C₁₀)-alkoxy, —(C₁-C₁₀)-alkyl,        —(C₆-C₂₂)-aryl, —(C₆-C₁₄)-aryl-(C₁-C₁₀)-alkyl, —NR²³R²⁴, and        —O—(C₁-C₁₀)-alkyl-O—(C₁-C₁₀)-alkyl-R²⁵, or wherein NR²³R²⁴ can        together with N be a 5-6-membered heterocyclic ring,    -   R²³ and R²⁴ are independently selected from the group consisting        of —(C₁-C₁₀)-alkyl, —(C₁-C₂₀)-aryl,        —(C₆-C₁₄)-aryl-(C₁-C₁₀)-alkyl,        —(C₁-C₆)-alkyl-[NH(CH₂)_(c)]_(d)—NR²⁶R²⁷,    -   R²⁵ is selected from the group consisting of —H, —OH, -halogen,        -amino, —(C₁-C₁₈)-alkylamino, —COOH, —CONH₂ and        —COO(C₁-C₄)-alkyl,    -   R²⁶ and R²⁷ are independently selected from the group consisting        from —H, —(C₁-C₆)-alkyl, and —(C₁-C₄)-alkoxy-(C₁-C₆)-alkyl    -   R²⁹ is selected from the group consisting of —OR³⁰ and —SR³⁰,    -   R³⁰ is selected from the group consisting of —H,        —(C₁-C₁₀)-alkyl, —(C₂-C₁₀)-alkenyl, —(C₆-C₂₂)-aryl, a protecting        group, a solid phase and a reporter group    -   B is the link to a moiety of formula I,    -   and any salts thereof.

For the definitions and preferences the particulars apply as outlinedfor the substituents under formulae I and II, if not specified otherwisespecifically for formula III.

Preferably, in compounds of formula II, R¹⁴ is hydrogen. Preferreddefinition of L is oxy. Preferred definition of U is —OH and —O-reportergroup. Preferred definition of V is oxy. Preferred definition of c is aninteger from 2 to 4, and of d an interger from 0 to 2.

Compounds of formula II are especially suited to contain theheterocyclic moiety of the invention as an integrated part (preferablynot at one of the termini) of the nucleic acid binding compound.

The group NR²³R²⁴ is preferably selected from the group consisting ofdialkylamino groups. In case of this group together with the forming of5- or 6-membered heterocyclic ring, it assumes preferably the definitionof pyrrolidinyl or piperidinyl.

Preferred aryl group is the phenyl or naphtyl moiety, eitherunsubstituted or substituted by one or more of amino, -aminoalkyl,—O—(C₁-C₁₀)-alkyl, —S—(C₁-C₁₀)-alkyl, —(C₁-C₁₀)-alkyl, sulfonyl,sulfenyl, sulfinyl, nitro and nitroso. Most preferred aryl group is thephenyl group. Preferred arylalkyl group is the benzyl group. Thepreferred alkylamino group is the ethylamino group. The preferred—COO(C₁-C₄) alkyl group contains one or two carbon atoms in the alkylmoiety (methyl or ethyl esters).

Nucleic acid binding compounds, wherein the group of formula I isattached to submit, for example the nucleotide, at the 3′-terminus ofthe compound, are useful either as starting compound for longercompounds or/and as end-labelled probes. This group of compounds isespecially preferred because the terminal position of probes generallyis the most tolerant in view of attachment of chemical moieties.

A preferred subject of the invention is a nucleic acid binding compoundas outlined above comprising a backbone moiety of the formula V

wherein

-   -   A is selected from the group consisting of O, S and        N—(C₁-C₆)-alkyl,    -   M′ is selected from the group consisting of oxy, sulfanediyl,        —NR²²—, —(C₁-C₁₀)-alkyl, or —O—(C₁-C₁₀)-alkyl-O—, and        —S—(C₁-C₁₀)-alkyl-O— and —NR²²—(C₁-C₆)-alkyl-O—,    -   R²² is selected from the group of —H, a protecting group, a        reporter group and —(C₁-C₁₀)-alkyl,    -   R¹⁴ is selected from the group consisting of —H, —OH,        —(C₁-C₁₀)-alkoxy, —(C₂-C₁₀)-alkenyloxy, —(C₂-C₁₀)-alkynyloxy,        -halogen, azido, —SH, —S—(C₁-C₆)-alkylmercapto and NH₂,    -   R¹⁶ is selected from the group consisting of —H, —(C₁-C₈)-alkyl,        —(C₂-C₁₈)-alkenyl, —(C₂-C₁₈)-alkynyl), —(C₂-C₁₈)-alkyl-carbonyl,        —(C₃-C₁₉)-alkenyl-carbonyl, —(C₃-C₁₉)-alkynyl-arbonyl, —(C₆-C₁₄)        -aryl-(C₁-C₈)-alkyl, a protective group or a compound of formula        IV

-   -   wherein    -   T is selected from the group consisting of oxo, thioxo and        selenoxo,    -   U is selected from the group consisting of —OH, —SH, —SeH,        —(C₁-C₁₀)-alkoxy, —(C₁-C₁₀)-alkyl, —(C₆-C₂₂)-aryl,        —(C₆-C₁₄)-aryl-(C₁-C₁₀)-alkyl, —NR²³R²⁴, and        —O—(C₁-C₁₀)-alkyl-O—(C₁-C₁₀)-alkyl-R²⁵, wherein NR²³R²⁴ can        together with N be a 5-6-membered heterocyclic ring,    -   R²³ and R²⁴ are independently selected from the group consisting        of —(C₁-C₁₀)-alkyl, —(C₁-C₂₀)-aryl,        —(C₆-C₁₄)-aryl-(C₁-C₁₀)-alkyl,        —(C₁-C₆)-alkyl-[NH(CH₂)_(c)]_(d)—NR²⁶R²⁷,    -   R²⁵ is selected from the group consisting of —H, —OH, -halogen,        -amino, —(C₁-C₁₈)-alkylamino, —COOH, —CONH₂ and        —COO(C₁-C₄)-alkyl,    -   R²⁶ and R²⁷ are independently selected from the group consisting        from —H, —(C₁-C₆)-alkyl, and —(C₁-C₄)-alkoxy-(C₁-C₆)-alkyl    -   R²⁹ is selected from the group consisting of —OR³⁰ and —SR³⁰,    -   R³⁰ is selected from the group consisting of —H,        —(C₁-C₁₀)-alkyl, —(C₂-C₁₀)-alkenyl, —(C₆-C₂₂)-aryl, a protecting        group, a solid phase and a reporter group, and    -   B is the link to a moiety of formula I,    -   wherein any alkyl, alkenyl and alkynyl can be substituted or        unsubstituted,    -   and any salts thereof.

Those compounds are compounds that can be used as 5′-terminally labelledprobes. Regarding the definitions of the substituents, the definitionsas given above apply if not indicated otherwise.

A very preferred compound is a compound of formula V, wherein M is O,R¹⁶ is H and R¹⁴ is selected from the group consisting of hydrogen andhydroxyl.

The backbone of the nucleic acid binding compound has the function tobear the base pairing heterocycles such that the compound can bind to anucleic acid having a complementary sequence. Preferably, the degree ofcomplementarity in the naturally occurring bases will be in the rangefrom 70% up to 100% in a stretch of bases in a region effecting binding,compared to the stretch of same length in the region of the nucleic acidto be bound. Deletions and insertions of subunits in each sequence willtherefor, in this calculation, be counted as gaps until the next fittingbase and thus reduce complementarity by as persony bases as the gapcontains.

Preferred backbone contains sugar-phosphate moieties. From these, deoxysugar containing backbones are further preferred.

Each moiety in the backbone bearing a moiety capable of base pairing toa nucleic acid of complementary sequence, including the moieties of theinvention, are termed a subunit. Compounds are known that have backbonesmixed of different kinds of subunits. Recently, a new kind ofnon-natural nucleic acid binding compounds was described. They aretermed Peptide Nucleic Acids (PNA), as they contain at least one peptidebond between the subunits (WO 92/20702). The nucleic acid bindingcompound of the present invention can have any length. However, due tothe convenience of chemical synthesis, compounds of a length of lessthan 100, more preferably from 10 to 30 subunits, for examplenucleosides, are preferred.

The nucleic acid binding compound of the present invention can beprepared analogous to known methods.

In a first option which is particularly suitable for short compounds,the compounds are produced by chemical synthesis (multistepoligomerisation) using chemically activated derivatives of the subunits(monomers), at least one of them containing the modified base of theinvention. Preferably, reactive groups in the monomers that are notinvolved in the actual reaction step of the oligomerisation reaction areprotected using an appropriate protecting group. Such protecting groupsare well known in the art and there will be no major change inprotection and synthesis strategy if the groups of the invention areused.

An activated subunit is a subunit containing a substituent especiallysuited for chemical reaction with a predetermined substituent in anothersubunit, on the surface of a solid phase or in an oligomer formed ofsubunits. Such especially suited subunits are preferably selected fromthe group consisting of phosphoramidites, phosphonates (likemethylphosphonates), phosphotriesters, phosphothioates,phosphodithioates, boranophosphates (see Chem. Commun. 1999, 1517-1518),phosphate methyl esters, phenylphosphonates and phosphate ethyl esters.

The predetermined substituents are preferably selected from the group of—NH₂, —SH and —OH.

A further subject of the invention is therefore a method for thechemical synthesis of a compound of any of claims 1 to 13 usingactivated subunits, wherein said subunit contains at least one group offormula I. There are several approaches known for the chemicalsynthesis, such as the phosphotriester method of Narang et al., Meth.Enzymol. 68, 90-99 (1979); the phosphodiester method of Brown et al.,Meth. Enzymol. 68, 109-151 (1979); the diethylphosphoramidite method ofBeaucage et al., Tetrahedron Lett. 22, 1859-1862 (1981); and the solidsupport method described in the U.S. Pat. Specification No. 4,458,066and in Methods in Molecular Biology, Ed. S. Agrawal, Vol. 20, HupersonaPress, Totowa, N.J., 1993. The most preferred method of chemicalsynthesis uses the phosphoramidite approach. A particularly preferredmethod uses a activated subunit one or more compounds of general formulaVII. This method has the advantage that it is very convenient and thereagents necessary, for example a phosphoramidite containing a group offormula I, is possible to be included easily.

A further subject of the invention are therefore compounds of thegeneral formula VII

-   -   wherein    -   A is selected from the group consisting of O, S and        N—(C₁-C₆)-alkyl,    -   M and M′ are independently selected from the group consisting of        oxy, sulfanediyl, —NR²², —(C₁-C₁₀)-alkyl, or        —O—(C₁-C₁₀)-alkyl-O—, and —S—(C₁-C₁₀)-alkyl-O— and        —NR²²—(C₁-C₆)-alkyl-O—,    -   R²² is selected from the group of —H and —(C₁-C₁₀)-alkyl,    -   R¹⁴ is selected from the group consisting of —H, —OR³¹,        —(C₁-C₁₀)-alkoxy, —(C₂-C₁₀)-alkenyloxy, —(C₂-C₁₀)-alkynyloxy,        -halogen, -azido NHR³¹, SR³¹ and —NH₂,    -   R³¹ is a protecting group or a reporter group,    -   R³² and R¹⁷ are independently selected from the group consisting        of —H, —(C₁-C₁₀)-alkyl, —(C₂-C₁₀)-alkenyl and —(C₆-C₂₂)-aryl,    -   R¹⁸ is selected from the group consisting of substituted or        unsubstituted —(C₁-C₆)-alkyl, unsubstituted —(C₁-C₆)-alkoxy or        —(C₁-C₆)-alkoxy substituted one or more times by a group        selected from the group consisting of -halogen, p-nitroaryloxy        and -cyano, and    -   B is a group of formula I.    -   Preferred compounds of formula VII are those wherein the group        of formula I is not 2-aza-hypoxanthine. In a preferred        embodiment, the group of formula I in formula VII contains at        least one reporter group. Most preferable, the group of formula        I contains exactly one reporter group.

Most preferred in such compounds, in —NR⁵R⁶ at least one of R⁵ and R⁶ isa protecting group.

Further subject of the invention are compounds of general formula IX

wherein

-   -   A is selected from the group consisting of O, S and        N—(C₁-C₆)-alkyl,    -   M and M′ are independently selected from the group consisting of        oxy, sulfanediyl, —NR²², —(C₁-C₁₀)-alkyl, or        —O—(C₁-C₁₀)-alkyl-O—, and —S—(C₁-C₁₀)-alkyl-O— and        —NR²²—(C₁-C₆)-alkyl-O—,    -   R²² is selected from the group of —H and —(C₁-C₁₀)-alkyl,    -   R¹⁴ is selected from the group consisting of —H, —OR³¹,        —(C₁-C₁₀)-alkoxy, —(C₂-C₁₀)-alkenyloxy, —(C₂-C₁₀)-alkynyloxy,        -halogen, -azido NHR³¹, SR³¹ and —NH₂,    -   R³¹ is a protecting group or a reporter group,    -   R³² and R¹⁷ are independently selected from the group consisting        of —H, —(C₁-C₁₀)-alkyl, —(C₂-C₁₀)-alkenyl and —(C₆-C₂₂)-aryl,    -   R¹⁸ is selected from the group consisting of substituted or        unsubstituted —(C₁-C₆)-alkyl, unsubstituted —(C₁-C₆)-alkoxy or        —(C₁-C₆)-alkoxy substituted one or more times by a group        selected from the group consisting of -halogen, p-nitroaryloxy        and -cyano, and    -   B is a group of formula I

-   -   wherein    -   W is selected independently from X, Y and Z from the group        consisting of N and CR²,    -   Z is selected from the group consisting of N and C with the        proviso that        -   if Z is N, then            -   X independently from W and Y is selected from the group                consisting of N and CR³, and            -   Y independently from W and X is selected from the group                consisting of N and CR⁴,            -   and the bond between X and Y is a double bond and the                bond between Y and Z is a single bond, and            -   if Z is C, then            -   X is NR³³, and            -   Y is selected from the group consisting of N and CR⁴ and            -   the bond between Z and Y is a double bond and the bond                between X and Y is a single bond,    -   R¹, R², R³ and R⁴ are independently selected from the group        consisting of —H, —halogen, —OR¹³, —SR¹⁹, —(C₁-C₁₀)-alkyl,        —(C₂-C₁₀)-alkenyl, —(C₂-C₁₀)-alkynyl, —NO₂, —NR⁵R⁶, -cyano, and        —C(═O)R¹¹,    -   R¹¹ is selected from the group consisting of —OH,        —(C₁-C₆)-alkoxy, —(C₆-C₂₂)-aryloxy, and NHR¹²,    -   R⁵, R⁶, R¹², R¹³, R¹⁹ and R³³ are selected independently from        the group consisting of —H, —(C₁-C₁₀)-alkyl, —(C₂-C₁₀)-alkenyl,        —(C₂-C₁₀)-alkinyl, —(C₆-C₂₂)-aryl, a protecting group and a        reporter group,    -   r and s are independently of each other an integer of 1 to 18,    -   D is the position of attachment of the group to the rest of the        nucleic acid binding compound, and    -   alkyl, alkenyl and alkynyl being unsubstituted or substituted by        one or more moieties selected from the group consisting of        -halogen, —S—(C₁-C₆)-alkyl, —(C₁-C₆)-alkoxy, —NR⁵R⁶, —CO—R¹¹,        —NH—CO—NR⁵R⁶, —NH—CSNR⁵R⁶— and —[O—(CH₂)_(r)]_(s)—NR⁵R⁶,    -   with the proviso that at least one of R⁵ and R⁶ of —NR⁵R⁶ is a        protecting group.

Those compounds can be used like those of formula VII in chemicalsynthesis.

A further subject of the invention are compounds of general formula X

-   -   wherein    -   M and M′ are independently selected from the group consisting of        oxy, sulfanediyl, —NR²², —(C₁-C₁₀)-alkyl, or        —O—(C₁-C₁₀)-alkyl-O—, and —S—(C₁-C₁₀)-alkyl-O— and        —NR²²—(C₁-C₆)-alkyl-O—,    -   R²² is selected from the group of —H and —(C₁-C₁₀)-alkyl,    -   R¹⁴ is selected from the group consisting of —H, —OR³¹,        —(C₁-C₁₀)-alkoxy, —(C₂-C₁₀)-alkenyloxy, —(C₂-C₁₀)-alkynyloxy,        -halogen, -azido NHR³¹, SR³¹ and —NH₂,    -   R³¹ is a protecting group or a reporter group,    -   B is a group of formula I

-   -   wherein    -   W is selected independently from X, Y and Z from the group        consisting of N and CR²,    -   Z is selected from the group consisting of N and C with the        proviso that        -   if Z is N, then            -   X independently from W and Y is selected from the group                consisting of N and CR³, and            -   Y independently from W and X is selected from the group                consisting of N and CR⁴,            -   and the bond between X and Y is a double bond and the                bond between Y and Z is a single bond, and            -   if Z is C, then            -   X is NR³³, and            -   Y is selected from the group consisting of N and CR⁴ and            -   the bond between Z and Y is a double bond and the bond                between X and Y is a single bond,    -   R¹, R², R³ and R⁴ are independently selected from the group        consisting of —H, -halogen, —OR¹³, —SR¹⁹, —(C₁-C₁₀)-alkyl,        —(C₂-C₁₀)-alkenyl, —(C₂-C₁₀)-alkynyl, —NO₂, —NR⁵R⁶, -cyano, and        —C(═O)R¹¹,    -   R¹¹ is selected from the group consisting of —OH,        —(C₁-C₆)-alkoxy, —(C₆-C₂₂)-aryloxy, and NHR¹²,    -   R⁵, R⁶, R¹², R¹³, R¹⁹ and R³³ are selected independently from        the group consisting of —H, —(C₁-C₁₀)-alkyl, —(C₂-C₁₀)-alkenyl,        —(C₂-C₁₀)-alkinyl, —(C₆-C₂₂)-aryl, a protecting group and a        reporter group,    -   r and s are independently of each other an integer of 1 to 18,    -   D is the position of attachment of the group to the rest of the        nucleic acid binding compound, and    -   alkyl, alkenyl and alkynyl being unsubstituted or substituted by        one or more moieties selected from the group consisting of        -halogen, —S—(C₁-C₆)-alkyl, —(C₁-C₆)-alkoxy, —NR⁵R⁶, —CO—R¹¹,        —NH—CO—NR⁵R⁶, —NH—CSNR⁵R⁶— and —[O—(CH₂)_(r)]_(s)—NR⁵R⁶.

Those compounds are also useful in chemical synthesis.

In another option which is more suited for long oligomers and thosebased on natural backbones, the oligomers are produced enzymatically. Inthis case, a starting oligomer is reacted with a polymerase and atriphosphate or modified triphosphate such that a monophoshate or amodified monophosphate is attached to a terminus of the oligomer, thuselongating the oligomer. Also for this method, the person skilled in theart will know several possible formates, like the nick-translationapproach, or the simple primer extension (J. Sambrook. E. F. Fritsch, T.Personiatis, Molecular Cloning—A laboratory Personual, Cold SpringHarbor Laboratory Press 1989).

For example, the incorporation of z²A_(d) into a DNA sequence can beperformed via conventional methods, e.g. by polymerase-catalyzedincorporation of Z²A_(d)-5′-triphosphate (11).

A further subject of the invention is therefore a method for theenzymatic synthesis of a nucleic acid binding compound according to theinvention comprising reacting a triphosphate subunit with a primer usinga nucleic acid as a template for the elongation of the primer, whereinthe triphosphate subunit contains a heterocyclic group of formula I.Preferably, the triphosphate subunit has the formula VI.

A further subject of the present invention are therefore compounds ofthe general formula VI

-   -   wherein    -   A is selected from the group consisting of O, S and        N—(C₁-C₆)-alkyl,    -   R¹⁴ is selected from the group consisting of —H, —OH,        —(C₁-C₁₀)-alkoxy, O-protecting group, S-protecting group,        NH-protecting group, —(C₂-C₁₀)-alkenyloxy, -halogen, -azido,        —SH, —(C₁-C₆)-alkylmercapto and —NH₂,    -   R¹⁵ and R¹⁶ are independently selected from the group consisting        of —H, —(C₁-C₈)-alkyl, —(C₂-C₁₈)-alkenyl, —(C₂-C₁₈)-alkynyl,        —(C₂-C₁₈)-alkyl-carbonyl, —(C₃-C₁₉)-alkenyl-carbonyl,        —(C₃-C₁₉)-alkynyl-carbonyl, —(C₆-C₁₄)-aryl-(C₁-C₈)-alkyl, a        protecting group or a compound of formula IV

-   -   wherein    -   T is selected from the group consisting of oxo, thioxo and        selenoxo,    -   U is selected from the group consisting of —OH, —SH, —SeH,        —(C₁-C₁₀)-alkoxy, —(C₁-C₁₀)-alkyl, —(C₆-C₂₂)-aryl,        —(C₆-C₁₄)-aryl-(C₁-C₁₀)-alkyl, —NR²³R²⁴, and        —O—(C₁-C₁₀)-alkyl-O—(C₁-C₁₀)-alkyl-R²⁵, or wherein NR²³R²⁴ can        together with N be a 5-6-membered heterocyclic ring,    -   R²³ and R²⁴ are independently selected from the group consisting        of —(C₁-C₁₀)-alkyl, —(C₁-C₂₀)-aryl,        —(C₆-C₁₄)-aryl-(C₁-C₁₀)-alkyl,        —(C₁-C₆)-alkyl-[NH(CH₂)_(C)]_(d)—NR²⁶R²⁷,    -   R²⁵ is selected from the group consisting of —H, —OH, -halogen,        amino, —(C₁-C₁₈)-alkylamino, —COOH, —CONH₂ and COO(C₁-C₄)-alkyl,    -   R²⁶ and R²⁷ are independently selected from the group consisting        from —H, —(C₁-C₆)-alkyl, and —(C₁-C₄)-alkoxy-(C₁-C₆)-alkyl,    -   R²⁹ is selected from the group consisting of —OR³⁰ and —SR³⁰,    -   R³⁰ is selected from the group consisting of —H,        —(C₁-C₁₀)-alkyl, —(C₂-C₁₀)-alkenyl, —(C₆-C₂₂)-aryl, a protecting        group, a diphosphate and a reporter group, and    -   M and M′ are independently selected from the group consisting of        oxy, sulfanediyl, —NR²², —(C₁-C₁₀)-alkyl, or        —O—(C₁-C₁₀)-alkyl-O—, and —S—(C₁-C₁₀)-alkyl-O— and        —NR²²—(C₁-C₆)-alkyl-O—,    -   R²² is selected from the group of —H and —(C₁-C₁₀)-alkyl, and    -   B is a moiety of formula I,    -   wherein any alkyl, alkenyl and alkynyl can be substituted or        unsubstituted, and wherein at least one of R¹⁵ and R¹⁶ is not a        group of formula IV with the proviso that    -   MR¹⁶, MR¹⁵ and R¹⁴ are not each —OH if R¹ is —NH₂ and if either    -   W and X and Y and Z is N, or    -   W and X and Z is N and Y is CR⁴, or    -   W and Y and Z is N and X is CR³.

Most preferred in these compounds —MR¹⁶ is a triphosphate group and—MR¹⁵ is OH. The most preferred compound is the one in which R¹⁴ is —OH.

Those compounds are especially the compounds, wherein the heterocyclicmoiety of the invention is contained not at the terminal position of thenucleic acid binding compound.

Preferred compounds are those, wherein M is oxy or sulfanediyl, R¹⁶ is acompound of formula IV wherein U is —OH, T is oxo or thioxo, R²⁹ is—OR³⁰ and R³⁰ is a disphosphate group and the salts thereof

Most preferred compounds are of formula VIII

-   -   wherein    -   PPP is a triphosphate group,    -   R¹⁴ is selected from the group consisting of —H, —OH,        —(C₁-C₁₀)-alkoxy, —(C₂-C₁₀)-alkenyloxy, —(C₂-C₁₀)-alkynyloxy        halogen, -azido and NH₂, and    -   B is a group of formula I,    -   with the proviso that R¹⁴ is not OH if B is 2-azaadenine.

In another preferred embodiment, the group of formula I in compounds offormula VII is selected from the group consisting of groups of formulaI, wherein either

-   -   W is N, Z is N, Y is N and X is CR³, or    -   W is N, Z is C, Y is N and X is CR³, or    -   W is N, Z is N, Y is N and X is N.

A further subject of the invention is a method for the enzymaticsynthesis of a nucleic acid binding compound of the invention comprisingreacting a triphosphate subunit with a primer using a nucleic acidtemplate for the elongation of the primer, wherein the triphosphatesubunit contains a heterocyclic group of formula I.

More preferable, this method uses as a triphosphate subunit a compoundof formula VIII as defined above.

The compounds of general formulae VI, VII and VIII can be prepared fromcompounds readily available. In a first embodiment, the compounds offormula VI characterized by the fact that the atom in the 2-position(purine numbering) are prepared by chosing the corresponding compoundhaving a carbon atom in this position (preferably wherein MR¹⁵ is OH andM′R¹⁶ is OH and R¹ is NH₂, reacting it with 2-chloroacetaldehyde,cleaving the pyrimidine ring using alkaline conditions and again closingthe ring using sodium nitrite and thereafter hydrolyzing the imidazolering introduced by the aldehyde. This reaction yields a replacement ofthe carbon atom of the 2-position by a nitrogen atom.

This compound can then be reacted with reagents to attach protectinggroups or/and activating groups, like phosphoramidites or phosphates, tothe hydroxyl groups intended to be used in oligomerization. Methods forattaching protecting groups, like the dimethoxytrityl group aregenerally known to a person skilled in the art. Also the attachment ofmono-, di- and triphosphate groups are known in the art.

In a preferred embodiment, the method for the preparation of a compoundof formula VII, wherein R¹ is a protected amino group, M′R³¹ isO-protecting group and MPR¹⁸NR³²R¹⁷ is O-phosphoramidite group, acompound of formula VI, wherein M′R¹⁶ and MR¹⁵ are each OH and R¹ is NH₂is subjected to conditions to protect the amino group, preferably by theprotecting group dialkylaminomethylidene, then reacted with reagents toprotect the 5′-hydroxyl group and then reacted with an activatedphosphane to produce the phosphoramidite. The resulting compound offormula VII can be used in the chemical synthesis for the introductionof the 2-aza-purine directly. The protecting group at R¹ will be removedduring the chemical synthesis.

For an alternative synthesis of 2-aza-2′-deoxyadenosine see N. Yamaji,M. Kato, Chemistry Lett. 1975, 311-314).

By the above methods, it is principally possible to introduce only onemonomer containing the moiety of the invention, but also more that one,as the case may be. The highest reduction of T_(m) will occur, if all Cin the binding region are replaced. This allows fine tuning of the Tm.Of course there may also be remaining Cs in any regions that are notintended to base pair with the nucleic acid to be determined.

Compounds of formula I bis VIII, in which any of substituents R¹, R², R³and R⁴ are selected from the group consisting of halogen, especially Cl,cyano or SR¹⁹ are useful as intermediate products for the synthesis ofcompounds wherein those substituents are selected from the group of—NR⁵R⁶ and —OR¹³, preferably —NR⁵R⁶. The intermediate products can beconverted into the final products by substitution reaction.

A further subject of the invention is a method for the determination ofa nucleic acid comprising the steps

-   -   providing a sample suspected to contain said nucleic acid,    -   providing a nucleic acid binding compound of claim 1, which is        essentially complementary to a part or all of said nucleic acid,    -   contacting said sample with said nucleic acid binding compound        under conditions for binding said nucleic acid binding compound        to said nucleic acid,    -   determining the binding product formed from said nucleic acid        and said nucleic acid binding compound as a measure of the        presence of said nucleic acid.

Methods for the determination of nucleic acids by hybridization aregenerally known in the art, for example from Sambrook et al. (citedabove). They can easily be adopted to the use of the probes of thepresent invention. Preferably, the nucleic acid binding compound will bebound to the nucleic acid in solution, as the reaction is faster than ona solid phase. It is apparent to a person skilled in the art how todetermine the Tm of the hybrid of the nucleic acid to be determined andthe probe prior to the construction of an assay and its general outset.If determined once, it should be clear to the person that he shouldchoose similar conditions in each assay using the same analyte nucleicacid.

Such determination will start with providing a sample which is suspectedto contain the nucleic acid to be determined. The sample may have beensubject to steps for bringing the nucleic acids in an appropriate form,for example by lysing any cells, in which the nucleic acids may becontained and would otherwise not be possible to be determined. Further,any steps to provide the sample could contain steps to purify thenucleic acids to be determined from components of an original samplethat could affect the determination. Such components could be enzymesthat would, if a nucleic acid is set free from cells, would digest ordegrade the nucleic acid, for example RNases. It is further preferred toalso remove from the nucleic acids components of the original samplethat could affect amplification of the nucleic acids.

There are several possibilities for methods for the determination ofnucleic acids using the nucleic acid binding compound of the invention.In a first group, the nucleic acid binding compound is used as adetectable probe. In this case, the nucleic acid binding compound willcontain a detectable reporter group. Any hybrids formed from the nucleicacid binding compound and a nucleic acid can then be determined via thedetectable reporter group. This group of assays can further be devidedinto two groups, one being the group of homogeneous assays and the otherbeing the heterogeneous assays. In heterogeneous assays, preferably thehybrid (binding product) will be determined when bound to a solid phase.This embodiment has the advantage that any excess of probe and othercomponents can be removed easily from the hybrid, thus make thedetermination easier. The hybrid formed can be captured to a solid phaseeither covalently, noncovalently, specifically or unspecifically. Thereare several embodiments which are known to a person skilled in the art.

In the so-called homogeneous assays, the hybrid formed will not be boundto a solid phase, but will be determined either directly or indirectlyin solution. A preferred example of such assays is disclosed in PCT/US91/05571 which is incorporated by reference here. The nucleic acidbinding compound of the invention is especially useful as a probe inthis assay. As the assay disclosed therein may need fine tuning ofmelting temperatures of the primers and probes used in these assays, thepresent invention using the modulation of a melting temperature of thehybrid formed by the nucleic acid to be determined or the amplificatesthereof and the probe is especially useful. This is especially usefulsince selectivity could be preserved, as the Tm is reduced by choosinga(n-x)-mer oligonucleotide instead of an n-mer oligonucleotide.

Therefore, a further subject of the invention is a method for thedetermination of the presence, absence or amount of a nucleic acid in asample comprising the steps:

-   -   providing primers, a first primer being essentially        complementary to a first binding sequence of said nucleic acid,        and the second primer being essentially complementary to a        binding sequence of a complement of this nucleic acid, and a        probe being complementary to the nucleic acid or the complement        thereof between the binding sequences of said primers, said        probe being labelled at different subunits by at least two        different reporter groups,    -   subjecting the sample with said primers and said probe under        conditions favouring extention of said primers and separating        said reporter groups from each other by disintegrating the        probe, and    -   determining the extent of disintegration of the probe via at        least one of said reporter groups,        wherein at least one of said primer or/and probe are a nucleic        acid binding compound as defined above. Preferably, the melting        point of the primers and the probe will be selected such that        the T_(m)'s are similar, but that the T_(m) of the probe is        higher than those of the primers.

In another embodiment, the nucleic acid binding compound of theinvention can be used as a immobilizable or immobilized probe forbinding any nucleic acids to a solid phase. Modes to immobilize thecompound are disclosed above. In an assay, it is either possible toimmobilize the compound before contacting it with the sample or duringcontacting the sample with a solid phase, or even after contacting thesample with the solid phase. In each of these cases, the nucleic acid tobe determined will be bound to the solid phase and preferably, anysubstituents of the sample not to be determined may be washed away fromthe solid phase, while the compounds and the nucleic acid will remainbound. Thereafter, the hybrid bound to the solid phase can be determinedby known methods, for example by using detectably labelled probe asoutlined above or by direct determination of the hybrid, for example bycontacting the hybrid with the intercallating dyes and measuring thechange on the solid phase.

In another embodiment, the immobilized probe is used to isolate orpurify a nucleic acid.

Another preferred embodiment uses a nucleic acid binding compound whichis both bound to a solid phase and labelled by a detectable reportergroup. The label in this case preferably is a group the detectableproperties of which will change when the nucleic acid to be determinedwill bind to the probe. Again, those compounds are known in the art.

The person skilled in the art will be in the position to design thesequence of a nucleic acid binding compound when knowing the sequence ofthe nucleic acid to which the nucleic acid binding compound is intendedto be bound. In almost all cases, the nucleic acid will have a sequencecontaining all four natural nucleic bases. In this case, it ispreferable to choose all bases that when bound to the particularsstretch of the nucleic acid are located at positions capable of basepairing to C, A and T of the nucleic acid to be G, T and A respectively.However, these bases can be replaced by equivalent bases base pairing tothe mentioned bases in the nucleic acid. The base being at the positioncapable of base pairing to G in the nucleic acid, will now be replacedone or more time in the nucleic acid binding compound by a group of I.As outlined in this invention, the group of I can also base pair toother moieties, for example to force the orientation of binding from theantiparallel (regular) mode to the parallel (non-natural) orientation.

A further subject of the present invention is the use of 2-azapurine ina nucleic acid binding compound as a substitute for cytosine, especiallyfor the binding of nucleic acids the bases of which are not onlyconsisting of G.

A further subject of the present invention is the use of 2-azapurine inhybridization reactions of probes with the nucleic acid as a base at theposition of the probe base pairing at G in the nucleic acid.

Further subject of the present invention is a binding product of atleast one nucleic acid binding compound of the invention and a nucleicacid, the nucleic acid binding compound and the nucleic acid being boundto each other by base pairing in parallel or antiparallel orientation.The binding product can contain one molecule of the nucleic acid and onemolecule of the nucleic acid binding compound, which form a duplex, orthe binding product can contain three strands, thus being a triplex. Thetriplex can contain either two molecules of the nucleic acid bindingcompound and one strand of the nucleic acid, or can contain one nucleicacid binding compound and two molecules of the nucleic acid. Which kindof triplex is formed, is dependent upon the concentration and thecomplementarity of the nucleic acid binding compound and the nucleicacid.

The present invention provides for the possibility to choose similar Tmsfor a number of probes with different sequence. Thus, a particularsubject of the invention are methods, wherein nucleic acids of differentsequences are to be isolated or determined simultaneously.

In a first embodiment, this method is a so-called multiplex method forthe isolation or determination of nucleic acids. In this embodiment,probes each containing a sequence complementary to a sequence of one ofthe nucleic acids (for example, nucleic acids from different viruses,like HCV, HIV and HBV) having a T_(m) adapted according to the invention(one or more probes containing a group of formula I) are contacted withthe sample suspected to contain said nucleic acids. Depending upon theformate, the different nucleic acids can be determined via the hybridsformed with the probes either as a sum or independently.

A second embodiment is based on arrays of probes. Especially, a subjectof the invention is a method for the determination of the presence orabsence of nucleic acids each comprising a particular sequence in asample comprising the steps

-   -   contacting said sample with a solid phase having immobilized on        its surface nucleic acid binding compounds each containing a        sequence complementary to one of said particular sequences of        said nucleic acids,    -   determining on said solid phase the formation of hybrids        containing a nucleic acid with a particular sequence and the        nucleic acid binding compound containing the complementary        sequence    -   characterized in that said at least one of said nucleic acid        binding compounds is a compound comprising a backbone, said        backbone having attached heterocyclic groups capable of base        pairing to natural nucleobases at least one of said heterocyclic        groups being one of the naturally occurring nucleobases        characterized in that at least one other of said heterocyclic        groups is a group of the general formula I.

Methods of this type are either useful when simultaneously testing asample for the presence or absence of nucleic acids, for example a panelof different bacterial species. In this way, it is not only possible totest for one species after each other batchwise, but to receive sequenceinformation on different nucleic acids simultaneously. Those types ofmethods are generally known in the art. The present invention, however,has found that the nucleic acid binding compounds of the invention areparticularly useful in such kind of assays, as the melting temperaturecan be adjusted to be very similar for nucleic acid binding compounds ofdifference sequence or/and length. It is evident that in order toachieve similar melting temperatures of each of the nucleic acid bindingcompounds, not all of these different compounds need to be modifiedaccording to the invention, but the person skilled in the art willmodify just those compounds which do not behave like the others, forexample by having a much higher melting temperature than the meltingtemperatures of the other compounds on the same solid phase. With each Creplaced according to the invention, the T_(m) may decrease by between 3and 6° C., preferably by between 4 and 5° C. In this respect, the personskilled in the art will, compared to the chip technology presentlyknown, has much more flexibility to select the particular sequences usedfor binding the different nucleic acids. For example, the high G-Ccontent of such regions in nucleic acids to be determined up to nowprevented these regions from being useful as target regions for suchassays based on probes complementary to these regions. According to thepresent invention, even these regions, which may be highly specific tothe nucleic acid to be determined, can now be used in such assays.

Assays using array technology are generally known to a person skilled inthe art, for example, from EP-A-0 476 014. Those solid phases arepreferably flat carriers. On their surface, separated by pure surface,arrays, each of the arrays having bound a probe of different sequencedirected to a specific particular sequence in a nucleic acid. Dependingupon the needs of the actual assay, the sequence of the probe will beessentially complementary to one (or more) of the particular sequencescontained in said nucleic acid to be determined. During the assay, eachnucleic acid will find the probe on the surface to which it can bind.The determination of the formation of hybrids in each array on the solidphase allows the determination of the presence or absence of nucleicacids containing the particular sequence based on the change of aproperty in this specific array. Preferably, the evaluation of thesechanges will be made assisted by a computer programme, which knows inwhich array which particular sequence is present. One chip can containfrom 2 to thousands, or even millions of arrays, each having bound anucleic acid binding compound having a specific sequence which may beunique. However, it is even possible to determine the presence of agroup of different nucleic acids, each having a sequence in common, justby using a nucleic acid binding compound being either relativelyunspecific and thus binding different sequences, or by selecting thesequence of the nucleic acid binding compounds such that it is directedto a sequence which is present on different nucleic acids.

In a second, slightly different approach, such arrays can be used todetermine the sequence of a nucleic acid by following the so-called“sequencing by hybridization” approach. In this mode, the samplepreferably will contain mostly nucleic acids of the same sequence. Thiscan be achieved by isolating the sequence from a mixture in which theywere contained or by enriching them in-vitro or in-vivo amplification.The sequence of the nucleic acid binding compounds bound to the solidphase will be selected such that they altogether contain a sequencecovering the sequence to be determined in the nucleic acid. Within thissequence, the sequences of each nucleic acid binding compound willoverlap by one or more bases. In the determination step of this method,it will be detected to which of these partial sequences the nucleic acidwill bind, which will only occur (ideally), if a sequence complementaryto the partial sequence is contained on a nucleic acid to be determined.Preferably, the evaluation of the result of the assay will be made by acomputer. The computer will thus determine from the partial sequencesthat are apparently contained in the sequence to be determined, in whichseries they must have been arranged in the whole nucleic acid.Especially in this kind of assays, the present invention is veryhelpful, as this approach needs a high number of nucleic acid bindingcompounds to be fixed on the surface, which can not always take intoaccount the problems occuring with high G-C content. In the presentinvention, it is therefore possible to sequence even nucleic acidshaving sequences of low and of high G-C content simultaneously.

It is apparent to a person skilled in the art that any compound asdisclosed herein can to some extent be present as tautomers and salts.These tautomers and salts, preferably the alkali salts, most preferredthe sodium salts, are within the definition of the formulae and subjectto the present invention.

The present invention is explained in more detail by the followingexamples:

Examples

General

Monomers: Flash chromatography (FC): at 0.5 bar with silica gel 60(Merck, Darmstadt, Gerpersony). Solvent systems for FC and TLC:CH₂Cl₂-MeOH 85:15 (A), EtOAc-MeOH 3:1 (B), CH₂Cl₂-MeOH 80:20 (C),CH₂Cl₂–HOAc–MeOH 17:1:3 (D), CH₂Cl₂—MeOH 9:1 (E), CH₂Cl₂-acetone 85:15(F). Samples were collected with an UltroRac II fractions collector (LKBInstruments, Sweden). Melting points: Büchi SMP-20 apparatus (Büchi,Switzerland). UV spectra: U-3200 spectrophotometer (Hitachi, Japan). NMRspectra: AC-250 and AMX-500 spectrometers (Bruker, Gerpersony); δ valuesare relative to internal Me₄Si or external H₃PO₄. Fluorescence spectrawere recorded in H₂O on a

F-4500 fluorescence spectrophotometer (Hitachi, Japan). Microanalyseswere performed by Mikroanalytisches Laboratorium Beller (Gottingen,Gerpersony).

Oligonucleotides: Oligonucleotides were synthesized with a ABI 392 DNAsynthesizer (Applied Biosystems, Gerpersony) according to the standardprotocol using the “trityl-off” mode, except for the unmodifiedoligodeoxynucleotides which were synthesized using the “trityl-on” mode.The coupling yields of modified phosphoramidites were generally 95% onaverage (trityl conductivity monitoring). The detritylated modifiedoligomers were purified by ion-exchange chromatography on a DionexNucleopac PA-100 HPLC column (4×250 mm, P/N 043010, Dionex GmbH,Idstein, Gerpersony) using the following gradient: 5 min 5% 0.01 MNaOH/1.5 M aq. LiCl (X) in 0.01 M NaOH (Y); 25 min 5-30% Y in X; 10 min30-5% Y in X; 5 min 5% Y in X. Ion-exchange HPLC apparatus: L-4250UV/VIS detector, L-6250 Intelligent pump and D-2500 integrator(Merck-Hitachi, Gerpersony). The tritylated unmodified oligonucleotideswere purified by RP-18 HPLC using the following apparatus and procedure:250×4 mm RP-18 column (Merck, Gerpersony); Merck-Hitachi HPLC apparatusconsisting of a 655 A-12 liquid chromatograph with a 655 A variablewavelength UV monitor and a D-2000 Chromato-Integrator (Merck-Hitachi,Darmstadt, Gerpersony); gradients of O.1 M (Et₃NH)OAc (pH 7.0)/MeCN 95:5(U) and MeCN (V); gradient I: 0-50 min 0-50% V in U, flow rate 1 mL/min;gradient II: 0-20 min 0-20% V in U; 20-40 min 20-40% V in U, flow rate 1mL/min. Detritylation was performed by treating the purified oligomerswith a 2.5% dichloroacetic acid solution in CH₂Cl₂ (1 mL) for 5 min.After neutralization with Et₃N, evaporation to dryness, followed byco-evaporation with MeOH, the oligomers were again purified by RP-18HPLC using the above-mentioned device. Gradient: 0-30 min 0-20% V in U,30-35 min 20% V in U, 35-40 min 20-0% V in U, 40-45 min 0% V in U.Subsequent desalting for all oligonucleotides was performed on an RP-19HPLC column (4×100 mm) using the apparatus as described above. Solventfor adsorption: H₂O, solvent for desorption: MeOH—H₂O 3:2. General flowrate: 1 mL/min. MALDI-TOF Mass spectra of the oligonucleotides weremeasured on a home-built apparatus using UV laser irradiation at 337 nmfor 3 nsec.

The enzymatic hydrolysis of the oligomers was performed as described inHelv. Chim. Acta 1998, 81, 1139-1155, but using a flow rate of 0.6mL/min. Quantification of the constituents was made on the basis of thepeak areas, which were divided by the extinction coefficients of thenucleoside (ε₂₆₀ values: dA 15400, dC 7300, dG 11400, dT 8800, z²A_(d)8200). Snake venom phosphodiesterase EC 3.1.15.1, Crotallus durissus)and alkaline phosphatase (EC 3.1.3.1, E. coli) used for the enzymatichydrolysis of oligonucleotides were from Roche Diagnostics GmbH.

Determination of melting curves and thermodynamics: Absorbance vs.temperature profiles were measured on Cary 1 or 1E spectrophotometers(Varian, Australia) with a Cary thermoelectrical controller. The T_(m)values were measured in the reference cell with a Pt-100 resistor, andthe thermodynamic data (ΔH°, ΔS°, ΔG°₂₉₈) were calculated with theprogram MeltWin 3.0. Circular dichroism (CD) spectra were recorded on aJasco 600 (Jasco, Japan) spectropolarimeter, a thermostaticallycontrolled bath (Lauda RCS-6) in a 1-cm cuvette.

Example 1

3-(2-Deoxy-β-D-erythro-pentofuranosyl)-3H-imidazo[2,1-i]purine(1,N⁶-Etheno-2′-deoxyadenosine, 5). 2′-Deoxyadenosine monohydrate (1)(5.0 g, 20 mmol) was dissolved in 1M aq. sodium acetate buffer (pH4.5-5.0, 110 mL) by warming to 40-50° C. To the solutionchloroacetaldehyde (50% aq. soln, 7.7 mol/L, 25 mL) was added, and thereaction mixture was stirred for 70 h at room temperature. The yellowsolution was evaporated to dryness, and the residue was dissolved inMeOH and filtered to remove inorganic salt. After washing with MeOH thecombined filtrate and washings were concentrated in vacuo at 40-50° C.The residue was applied to FC (silica gel 60H, column: 20×6 cm). Elutionwith CH₂Cl₂-MeOH (85:15) gave a main fraction from which uponevaporation of the solvent and subsequent crystallization fromMeOH-EtOAc compd. 5 (3.86 g, 70%) was isolated as colorless crystals.M.p. 138-141° C. TLC (silica gel, EtOAc-MeOH, 3:1): R_(f) 0.4. UV(MeOH): λ_(max) 275 (7300), 265 (7600), 258 (6600), 229 nm (35700).¹H-NMR ([D₆]DMSO) δ 2.39 (m, 1H, H_(α)—C(2′)); 2.70 (m, 1H,H_(β)—C(2′)); 3.57 (m, 1H, H_(a)—C(5′)); 3.67 (m, 1H, H_(b)—C(5′)); 3.88(m, 1H, H—C(4′); 4.43 (m, 1H, H—C(3′)); 4.99 (t, 1H, ³J(H,H)=5.2 Hz,5′-OH); 5.38 (d, 1H, ³J(H,H)=3.8 Hz, 3′-OH); 6.47 (pt, 1H, ³J(H,H)=6.2Hz, H—C(1′)); 7.55 (s, 1H, H—C(11); 8.07 (s, 1H, H—C(10); 8.53 (s, 1H,H—C(2)); 9.29 (s, 1H, H—C(8)).

Example 2

1-(2-Deoxy-β-D-erythro-pentofuranosyl)-5-amino-4-(imidazol-2″-yl)-imidazole(6). Compound 5 (3.85 g, 14 mmol) was treated with 1N aq. NaOH (60 mL)at room temperature overnight. The reaction mixture was adjusted to pH 7by addition of 2N aq. HCl and concentrated to a syrup. This wasdissolved in absolute MeOH, and the precipitated NaCl was filtered ofand washed with MeOH. Filtrate and washings were combined andevaporated. The residue was applied to FC (silica gel 60H, column: 20×6cm). Elution with CH₂Cl₂-MeOH (C) afforded a main zone from whichcompound 6 (2.70 g, 73%) was obtained as a colorless foam which was usedfor the next reactions without further purification. An analyticalsample was crystallized from MeOH-EtOAc to give colorless sphericalcrystals; m.p. 91-93° C. (decomp.). TLC (silica gel, CH₂Cl₂—HOAc-MeOH,17:1:3): R_(f) 0.22. Uv (MeOH): λ_(max) 271 nm (12800). ¹H-NMR([D₆]DMSO) δ 2.21 (m, 1H, H_(α)—C(2′)); 2.47 (m, 1H, H_(⊕)—C(2′)); 3.57(m, 2H, H₂—C(5′)); 3.84 (m, 1H, H—C(4′)); 4.36 (m, 1H, H—C(3′)); 6.00(pt, 1H, ³J(H,H)=6.5 Hz, H—C(1′)); 6.60 (br. s, NH₂); 7.13 (s, 2H,H—C(4)+H—C(5)); 7.55 (s, 1H, H—C(2)); 8.16 (s, NH).

Example 3

3-(2-Deoxy-β-D-erythro-pentofuranosyl)-1H-diimidazo[1,2-c:4′,5′-e][1,2,3]-triazine(1,N⁶-etheno-2-aza-2′-deoxyadenosine, 7). A solution of compound 6 (4.50g, 17 mmol) in 80% aq. HOAc was treated with sodium nitrite (1.17 g, 17mmol) in an ice-water bath for 1 h. The reaction mixture was evaporatedto a syrup. This was dissolved in H₂O and evaporated repeatedly toremove HOAc. The residue was applied to FC (silica gel 60H, column, 20×6cm). Elution with CH₂Cl₂-MeOH (85:15) afforded compound 7 (2.50 g, 53%)upon evaporation. M.p. 151-152° C. (decomp.). TLC (silica gel,CH₂Cl₂-MeOH, 4:1): R_(f) 0.5. UV (MeOH): λ_(max) 282 (3100), 268 (3200),238 nm (37900). ¹H-NMR ([D₆]DMSO) δ 2.54 (m, 1H, H_(α)—C(2′)); 2.85 (m,1H, H_(β)—C(2′)); 3.97 (m, 2H, H₂—C(5′)); 4.00 (m, 1H, H—C(4′)); 4.50(m, 1H, H—C(3′)); 4.96 (t, 1H, ³J(H,H)=5.4 Hz, 5′-OH); 5.41 (d, 1H,³J(H,H)=4.3 Hz, 3′-OH); 6.69 (pt, 1H, ³J(H,H)=6.3 Hz, H—C(1′)); 7.85 (d,1H, ³J(H,H)=1.1 Hz, H—C(11)); 8.75 (d, 1H, ³J(H,H)=1.1 Hz, H—C(10));8.95 (s, 1H, H—C(8)). Anal. calcd. for C₁₁H₁₂N₆O₃ (276.25): C 47.83, H4.38, N 30.42; found: C 47.71, H 4.32, N 30.32.

Example 4

4-Amino-7-(2-deoxy-β-D-erythro-pentofuranosyl)-7H-imidazo[4,5-d][1,2,3]-triazine(2-aza-2′-deoxyadenosine, 2). Compound 7 (0.56 g, 2 mmol) was dissolvedin 1M aq. sodium acetate buffer (pH 4.0-4.5, 120 mL) by warming to40-50° C. To this solution N-bromosuccinimide (2.8 g, 16 mmol) wasadded, and the reaction mixture was stirred at room temperatureovernight. The reaction mixture was evaporated and applied to a Dowex1×8 ion exchange column (3×12 cm, OH⁻ form). Elution with H₂O (250 mL)gave compound 2 (0.19 g, 38%) as colorless needles which decompose above185° C. The reaction product was identical with an authentic sample inall respects^([21]).

Example 5

7-(2-deoxy-β-D-erythro-pentofuranosyl)-7H-imidazo[4,5-d][1,2,3]-triazin-4-one(2-aza-2′-deoxyinosine, 3). Compound 2 (19 mg, 0.076 mmol) was dissolvedin H₂O and adenosine deaminase (2 μg, from calf intestine, dissolved inglycerole) was added. The reaction mixture was stirred for 18 h at roomtemperature until 2 had completely disappeared (UV monitoring) and thenevaporated to dryness in a SpeedVac concentrator. UV (H₂O): λ_(max) 247(5500), 290 nm (6200). ¹H-NMR (D₂O): 2.49 (m, 1H, H_(α)—C(2′)); 2.75 (m,1H, H_(β)—C(2′)); 3.41, 3.50 (2m, 2H, H₂—C(5′)); 4.03 (m, 1H, H—C(4′));4.51 (m, 1H, H—C(3′)); 6.43 (pt, 1H, ³J(H,H)=3.2 Hz, H—C(1′)); 8.31 (s,1H, H—C(8)).

Example 6

4-(Benzoylamino)-7-(2-deoxy-β-D-erythro-pentofuranosyl)-7H-imidazo[4,5-d][1,2,3]-triazine(8). Compound 2 (125 mg, 0.5 mmol) was co-evaporated twice withanhydrous pyridine. The residue was suspended in anhydrous pyridine andtreated with trimethylsilyl chloride (0.5 mL, 4 mmol). After few minutesof stirring a clear solution was formed. The reaction mixture wasstirred at room temperature for 2 h. Next, benzoyl chloride (0.25 mL, 2mmol) was added, and stirring was continued for another 2 h. Thereaction mixture was cooled in an ice-water bath, and H₂O (1 mL) wasadded. After 10 min the reaction mixture was treated with aqueous conc.NH₃ (0.8 mL) and left for additional 30 min. The mixture was thenevaporated to dryness, treated with H₂O, and extracted with EtOAc (3×20mL). The combined extracts were dried (Na₂SO₄) and applied onto a silicagel column (3×15 cm). Elution was performed with CH₂Cl₂ (150 mL),followed by CH₂Cl₂-MeOH (9:1). The nucleoside-containing fractions wereevaporated to dryness, and compound 8 was crystallised from MeOH—H₂O togive colorless needles (135 mg, 76%). M.p. 208-210° C. (decomp>170° C.).TLC (silica gel, CH₂Cl₂-MeOH 9:1): R_(f) 0.31. UV: (10% MeOH in water):λ_(max) 233 (15600), 276 nm (16400). ¹H-NMR ([D₆] DMSO): δ 2.97 (2m, 2H,H₂—C(2′)); 3.69 (m, 2H, H₂—C(5′)); 4.00 (m, 1H, H—C(4′)); 4.57 (m, 1H,H—C(3′)); 5.07 (t, 1H, ³J(H,H)=4.8 Hz, 5′-OH); 5.49 (d, 1H, ³J(H,H)=4.0Hz, 3′-OH); 6.72 (t, 1H, ³J(H,H)=6.5 Hz, H—C(1′)); 7.61-7.78 (m, 4H,aromatic-H), 8.16 (d, 2H, aromatic-H); 9.09 (s, 1H, H—C(8)); 11.84 (s,1H, N—H). Anal. calcd. for C₁₆H₁₆N₆O₄ (356.3): C 52.93, H 4.41, N 23.38;found: C 52.68, H 4.39, N 23.07.

Example 7

7-(2-Deoxy-β-D-erythro-pentofuranosyl)-4-{[(dimethylamino)methylidene]-amino}-7H-imidazo[4,5-d][1,2,3]-triazine(9a). To a stirred suspension of compound 2 (63 mg, 0.25 mmol) in MeOH(5 mL) N,N-dimethylformamide dimethylacetal (120 mg, 0.5 mmol) wasadded. Stirring was continued for 2 h at room temperature. The reactionmixture was evaporated to dryness, and the residue was adsorbed onsilica gel. Flash chromatography on a silica gel column (3×10 cm) withCH₂Cl₂ (100 mL) followed by CH₂Cl₂-MeOH (9:1) afforded colorless needles(MeOH—H₂O, 65 mg, 85%). M.p. 173-175° C. TLC (silica gel, CH₂Cl₂-MeOH,9:1): R_(f) 0.28. UV: (10% MeOH in H₂O): λ_(max) 234 (13250), 319 nm(29500). ¹H-NMR ([D₆] DMSO): δ 2.84 (2m, 2H, H—C(2′)); 3.17, 3.25 (2s,2H, N—CH₃); 3.60 (m, 2H, H₂—C(5′)); 3.92 (m, 1H, H—C(4′)); 4.47 (m, 1H,H—C(3′)); 5.05 (t, 1H, ³J(H,H)=4.9 Hz, 5′-OH); 5.39 (d, 1H, ³J(H,H)=4.0Hz, 3′-OH); 6.55 (t, 1H, ³J(H,H)=6.6 Hz, H—C(1′)); 8.79 (s, 1H, N═CH);9.08 (s, 1H, H—C(8)). Anal. calcd. for C₁₂H₁₇N₇O₃ (307.3): C 46.90, H5.58, N 31.90; found: C 46.55, H 5.68, N 31.66.

Example 8

7-(2-Deoxy-β-D-erythro-pentofuranosyl)-4-{[(di-isobutylamino)methylidene]-amino}-7H-imidazo[4,5-d][1,2,3]-triazine(9b). As described for 9a but using N,N-di-isobutylformamidedimethylacetal. Colorless crystals (72%). M.p. 138-140° C. TLC (silicagel, CH₂Cl₂-MeOH, 9:1): R_(f) 0.40. UV (10% MeOH in water): λ_(max) 236(10100), 325 nm (25850). ¹H-NMR ([D₆]DMSO): δ 0.88, 0.94 (2d, 12 H,CH₃); 1.95, 2.20 (2m, 2H, CH); 2.80 (2m, 2H, H₂—C(2′)); 3.30-3.74 (m,6H, H₂—C(5′) and 2 CH₂); 4.00 (m, 1H, H—C(4′)); 4.45 (m, 1H, H—C(3′));5.03 (t, 1H, ³J(H,H)=4.9 Hz, 5′-OH); 5.37 (d, 1H, ³J(H,H)=4.0 Hz,3′-OH); 6.54 (t, 1H, ³J(H,H)=6.4 Hz, H—C(1′)); 8.78 (s, 1H, N═CH); 9.10(s, 1H, H—C(8)). Anal. calcd. for C₁₈H₂₉N₇O₃.½ H₂O (400.5): C 53.99, H7.55, N 24.48; found: C 53.65, H 7.62, N 24.11.

Example 9

7-(2-Deoxy-β-D-erythro-pentofuranosyl)-4-{[(di-n-butylamino)methylidene]-amino}-7H-imidazo[4,5-d][1,2,3]-triazine(9c). As described for 9a but using N,N-di-n-butylformamidedimethylacetal. Colorless needles (75%). M.p. 107-109° C. TLC (silicagel, CH₂Cl₂-MeOH, 9:1): R_(f) 0.42. UV: (10% MeOH in water): λ_(max) 235(10200), 325 nm (25700). ¹H NMR ([D₆]DMSO): δ 0.93 (t, 6H, CH₃), 1.33,1.64, 3.70 (3m, 12H, —CH₂—); 2.45, 2.80 (2m, 2H, H₂—C(2′)); 3.60 (m, 2H,H₂—C(5′)); 3.92 (m, 1H, H—C(4′)); 4.48 (m, 1H, H C(3′)); 5.04 (t, 1H,³J(H,H)=5.8 Hz, 5′-OH); 5.39 (d, 1H, ³J(H,H)=4.0 Hz, 3′-OH); 6.55 (pt,1H, ³J(H,H)=6.3 Hz, H—C(1′)); 8.78 (s, 1H, N═CH); 9.08 (s, 1H, H—C(8)).Anal. calcd. for C₁₈H₂₉N₇O₃ (391.5): C 55.23, H 7.47, N 25.05; found: C55.36, H 7.66, N 24.97.

Example 10

7-[2-Deoxy-5-O-(4,4′-dimethoxytriphenylmethyl)-β-D-erythro-pentofuranosyl]-4-{[(di-n-butylamino)methylidene]amino}-7H-imidazo[4,5-d][1,2,3]-triazine(10a). Compound 9c (390 mg, 1 mmol) was co-evaporated twice withpyridine, and the oily residue was dissolved in anhydrous pyridine (6mL). Next, 4,4′-dimethoxytriphenyl-methyl chloride (450 mg, 1.3 mmol)was added, and the reaction mixture was stirred at room temperature for2 h. Thereupon, MeOH (0.2 mL) was added, and stirring was continued for15 min. The reaction mixture was poured into 15 mL of an aq. 5% NaHCO₃solution, and this was extracted twice with CH₂Cl₂ (30 mL, each). Thecombined extracts were dried over Na₂SO₄, evaporated, and the residuewas adsorbed on silica gel. This was applied onto a silica gel60H—column (4×14 cm) and chromatographed with a CH₂Cl₂-acetone gradient(0→25% of acetone, total volume, 600 mL). The nucleoside-containingfractions were pooled and evaporated to obtained compound 10a as solidfoam (560 mg, 81%). TLC: (silica gel, CH₂Cl₂-acetone, 85:15): R_(f)0.15. ¹H-NMR ([D₆] DMSO): 0.93 (t, 6H, CH₃); 1.34, 1.63, 3.75 (3m, 12H,—CH₂—); 2.95 (2m, 2H, H—C(2′)); 3.51 (m, 2H, H₂—C(5′)); 3.63, 3.69 (2s,6H, OCH₃); 4.01 (m, 1H, H—C(4′)); 4.59 (m, 1H, H—C(3′)); 5.45 (d, 1H,³J(H,H)=4.1 Hz, 3′-OH); 6.57 (pt, 1H, ³J(H,H)=6.2 Hz, H—C(1′));6.60-7.30 (m, 13H, phenyl-H); 8.71 (s, 1H, N═CH), 9.07. (s, 1H, H—C(8)).Anal. calcd. for C₃₉H₄₇N₇O₅ (693.8): C 67.51; H 6.83; N 14.13; found: C67.15, H 6.82, N 14.13.

Example 11

7-[2-Deoxy-5-O-(4,4′-dimethoxytriphenylmethyl)-β-D-erythro-pentofuranosyl]-4-{[(di-n-butylamino)methylidene]amino}-7H-imidazo[4,5-d][1,2,3]-triazine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] (10b). To a solution of compound 10a (300 mg, 0.43mmol) in anhydrous CH₂Cl₂ (20 mL) N,N-diisopropylethylamine (145 μL,0.88 mmol) and chloro-(2-cyanoethoxy)-N,N-diisopropylaminophosphine (143μL, 0.62 mmol) were added under an Ar atmosphere. After stirring for 20min at room temperature, a 5% aq. NaHCO₃ solution (15 mL) was added andthe mixture was extracted with CH₂Cl₂ (2×30 mL). The organic layer wasdried (Na₂SO₄), filtered and evaporated. FC (silica gel, column 5×10 cm,CH₂Cl₂/acetone, 85:15) gave a mixture of diastereoisomers of the titlecompound (300 mg, 78%). TLC (silica gel, CH₂Cl₂/acetone, 85:15): R_(f)0.71, 0.80. ³P-NMR (CDCl₃): 149.962, 150.223.

Example 12

5′-O-(4,4′-dimethoxytrityl)-6-N-((di-n-butylamino)methylene)-2-aza-2′-deoxyadenosine3′-[(2-Cyanoethyl)-N,N-(diisopropyl)]phosphoramidite (12) To a solutionof5′-O-(4,4′-dimethoxytrityl)-6-N-((di-n-butylamino)methylene)-2-aza-2′-deoxyadenosine(300 mg, 0.43 mmol) in anh. CH₂Cl₂ (20 ml) (i-Pr)₂EtN (145 μl, 0.88mmol) and chloro-(2-cyanoethoxy)(diisopropylamino)phosphine (143 μl,0.62 mmol) were added. After stirring for 20 min at r. t., a 5% aq.NaHCO₃ solution (15 ml) was added and the mixture was extracted withCH₂Cl₂ (2×30 ml). The organic layer was dried (Na₂SO₄), filtered andevaporated. FC (silica gel, column 5×10 cm, CH₂Cl₂/acetone, 85:15) gavea mixture of diastereoisomers NR-411 (300 mg, 77%). TLC (silica gel,CH₂Cl₂/acetone, 85:15): R_(f) 0.71, 0.80. ³¹P-NMR (CDCl₃): 149.962,150.223.

Example 13

Synthesis of nucleic acid binding compounds using the monomers ofExample 12

The synthesis was performed as outlined in the General section.

Example 14

Determination of nucleic acids using probes according to Example 13

TABLE 1 T_(m)-Values and Thermodynamic Parameters of Duplex Formation ofOligonucleotides Containing 2-Aza-2′-deoxyadenosine. SEQ ID T_(m) ΔH ΔSΔG Oligonucleotide NO: [° C.] [kcal/mol] [cal/K mol] [kcal/mol]5′-d(TAGGTCAATACT) 1 46 −82 −230 −10.4 3′-d(ATCCAGTTATGA) 25′-d(TAGGTC6ATACT) 3 42 −85 −245 −9.2 3′-d(ATCCAGTTATGA) 25′-d(TAGGTC6ATACT) 3 46 −83 −236 −9.9 3′-d(ATCCAGGTATGA) 45′-d(TAGGTC66TACT) 5 37 −76 −219 −7.7 3′-d(ATCGAGTTATGA) 25′-d(TAGGTC66TACT) 5 25 −49 −141 −5.7 3′-d(ATCCAGAAATGA) 65′-d(TAGGTC66TACT) 5 20 −41 −113 −5.5 3′-d(ATCCAGCCATGA) 75′-d(TAGGTC66TACT) 5 46 −74 −206 −10.1 3′-d(ATCCAGGGATGA) 85′-d(TAGGTGAATACT) 1 36 −.47 −127 −7.4 3′-d(ATCCAGGGATGA) 85′-d(TAGGTCGGTACT) 9 54 3′-d(ATCCAGCGATGA) 7 10 mM Na-cacodylate, 100 mMNaCl, 10 mM MgCl₂, pH 7; 5 μM single strand concentration; 6: z²A_(d).

The two oligonucleotides 5′-d(TAGGTCAATACT) (SEQ ID NO: 1) and5′-d(AGTATTGACCTA) (SEQ ID NO: 10) were constructed to form a stablehybrid with a T_(m) value of 47° C. This duplex is used as a standard tostudy the influence of modified bases on the duplex structure andstability. As can be seen from Table 1, the replacement of one centraldA-dT by a z²A_(d)-dT base pair reduces the T_(m) of the duplex by 5°;the exchange of two base pairs reveals a decrease of the T_(m) by 10°.The reduction of duplex stability is obviously independent from theposition of base pair replacement: the duplex having two consecutivez²A_(d)-dT pairs, exhibits the same T_(m) as the duplex in which themodified base pairs are separated by three regular ones. Duplexstability is linearly decreased further when the number of z²A_(d)-dTbase pairs is increased; the oligonucleotide containing four modifiedpairs exhibits a T_(m) of only 28° C. This result is in strikingcontrast to findings on oligonucleotides in which dA residues arereplaced by 8-aza-2′-deoxyadenosine; here, the introduction of even fourz⁸A_(d) residues instead of dA does not exert any influence on theduplex stability.

As can be seen from table 1, the TM of an oligonucleotide having atposition 7 a G-C base pair (last line in table 1) has a TM of 54° C.Replacement of two C's in these base pairs by 2-azaadenine yields a TMof 46° C. (7 duplex). The same TM is present when these artificial basepairs are replaced by the natural base pair A-T (first duplex).Moreover, the duplex is having mixed 2-azaadenine/G and A-T base pair(third duplex). From table 1 it can be learned that replacement of oneG-C base pair by an artificial base pair of the present inventionreduces the TM by between 3 and 5° C., preferably 4° C.

The above described results were found for duplexes with antiparallelchain orientation. The same result, however, was also found foroligonucleotide duplexes with a parallel strand polarity. The chainorientation of naturally-occuring DNA is antiparallel (aps). Thisorientation can be turned to parallel when the duplex containsisoG_(d)-dC and or isoMe⁵C_(d)-dG base pairs (Helv. Chim. Acta. 1997,80, 73-85). As the pairing of dA with dT is ambiguous any natural DNAcan be hybridized in the parallel mode when the second strand containsthe bases isoguanine, isocytosine, adenine and thymine. As an example,one duplex is given in Table 1. When in this duplex two dA-dT base pairsare replaced by z²A_(d)-dT, a reduction of the T_(m) value by 10° isdetermined which is identical with the results for correspondingantiparallel oligonucleotide duplexes.

The T_(m) data listed in Table 1 display another interesting feature ofthe base pairing properties of z²A_(d) (2). Stimulated by the findingthat replacement of a destabilizing central z²A_(d)-dT base pair byz²A_(d)-dG enhances the T_(m) value of the oligomer back to the value ofthe unmodified duplex (T_(m) 46° C.), we investigated the duplexstabilities of oligomers containing mismatches.

For this purpose oligodeoxynucleotides were synthesized in which twocentral z²A_(d) residues are placed opposite to either two dT, dA, dC ordG residues. In all cases, except for z²A_(d)-dG—containing duplexes,the T_(m) value is significantly decreased—most pronounced for theoligomer with two z²A_(d)-dC pairs. This oligonucleotide, however,exhibits almost the same value as the unmodified duplex. This promptedus to propose a z²A_(d)-dG base pair as shown in FIG. 1 and FIG. 6(motif I). Thus, the present invention can also be used in assays wherenucleic acids should be discriminated using mismatches.

The findings on the peculiar base pairing of 2-aza-2′-deoxyadenosineimply that this nucleoside exhibits similar pairing properties as2′-deoxyisoguanosine (isoG_(d)), the more so as both show a similarhydrogen bonding donor-acceptor pattern when assuming a keto/H—N(3)tautomeric form of 2′-deoxyisoguanosine (FIG. 6, motif II). Indeed, thelatter forms a purine-purine base pair with 2′-deoxyguanosine inoligodeoxynucleotides with an antiparallel strand polarity butsignificantly weaker base pairs with dC and dT, and particularly withdA.

With the aid of these results we anticipate that in parallel orientedoligonucleotides 2-aza-2′-deoxyadenosine will form a base pair withisoG_(d) (FIG. 7, motif III) under neutral conditions as well as withprotonated dC (FIG. 7, motif V) in antiparallel arranged duplexes. Onthe other hand, with protonated 2′-deoxyisocytidine an antiparallel basepair should be formed following the structural motif VI depicted in FIG.7.

1. A compound of formula VI

wherein A is selected from the group consisting of O, S andN—(C₁-C₆)-alkyl, R¹⁴ is selected from the group consisting of —H, —OH,—(C₁-C₁₀)-alkoxy, O-protecting group, S-protecting group, NH-protectinggroup, —(C₂-C₁₀)-alkenyloxy, -halogen, -azido, —SH,—(C₁-C₆)-alkylmercapto and —NH₂, R¹⁵ and R¹⁶ are independently selectedfrom the group consisting of —H, —(C₁-C₈)-alkyl, —(C₂-C₁₈)-alkenyl,—(C₂-C₁₈)-alkynyl, —(C₂-C₁₈)-alkyl-carbonyl, —(C₃-C₁₉)-alkenyl-carbonyl,—(C₃-C₁₉)-alkynyl-carbonyl, —(C₆-C₁₄)-aryl-(C₁-C₈)-alkyl, a protectinggroup or a compound of formula IV

wherein T is selected from the group consisting of oxo, thioxo andselenoxo, U is selected from the group consisting of —OH, —SH, —SeH,—(C₁-C₁₀)-alkoxy, —(C₁-C₁₀)-alkyl, —(C₆-C₂₂)-aryl,—(C₆-C₁₄)-aryl-(C₁-C₁₀)-alkyl, —NR²³R²⁴, and—O—(C₁-C₁₀)-alkyl-O—(C₁-C₁₀)-alkyl-R²⁵, or wherein NR²³R²⁴ can togetherwith N be a 5-6-membered heterocyclic ring, R²³ and R²⁴ areindependently selected from the group consisting of —(C₁-C₁₀)-alkyl,—(C₁-C₂₀)-aryl, —(C₆-C₁₄)-aryl-(C₁-C₁₀)-alkyl,—(C₁-C₆)-alkyl-[NH(CH₂)_(C)]_(d)—NR²⁶R²⁷, R²⁵ is selected from the groupconsisting of —H, —OH, -halogen, amino, —(C₁-C₁₈)-alkylamino, —COOH,—CONH₂ and COO(C₁-C₄)-alkyl, R²⁶ and R²⁷ are independently selected fromthe group consisting from —H, —(C₁-C₆)-alkyl, and—(C₁-C₄)-alkoxy-(C₁-C₆)-alkyl, R²⁹ is selected from the group consistingof —OR³⁰ and —SR³⁰, R³⁰ is selected from the group consisting of —H,—(C₁-C₁₀)-alkyl, —(C₂-C₁₀)-alkenyl, —(C₆-C₂₂)-aryl, a protecting group,a diphosphate and a reporter group, and M and M′ are independentlyselected from the group consisting of oxy, sulfanediyl, —NR²²,—(C₁-C₁₀)-alkyl, or —O—(C₁-C₁₀)-alkyl-O—, and —S—(C₁-C₁₀)-alkyl-O— and—NR²²—(C₁-C₆)-alkyl-O—, R²² is selected from the group of —H and—(C₁-C₁₀)-alkyl, and B is a moiety of formula I,

wherein any alkyl, alkenyl and alkynyl can be substituted orunsubstituted, and wherein at least one of R¹⁵ and R¹⁶ is not a group offormula IV with the proviso that MR¹⁶, MR¹⁵ and R¹⁴ are not each —OH ifR¹ is —NH₂ and if either W and X and Y and Z is N, or W and X and Z is Nand Y is CR⁴, or W and Y and Z is N and X is CR³.
 2. A compound offormula VII

wherein A is selected from the group consisting of O, S andN-(C₁-C₆)-alkyl, M and M′ are independently selected from the groupconsisting of oxy, sulfanediyl, —NR²², —(C₁-C₁₀)-alkyl, or—O—(C₁-C₁₀)-alkyl-O—, and —S—(C₁-C₁₀)-alkyl-O— and—NR²²—(C₁-C₆)-alkyl-O—, R²² is selected from the group of —H and—(C₁-C₁₀)-alkyl, R¹⁴ is selected from the group consisting of —H, —OR³¹,—(C₁-C₁₀)-alkoxy, —(C₂-C₁₀)-alkenyloxy, —(C₂-C₁₀)-alkynyloxy, -halogen,-azido NHR³¹, SR³¹ and —NH₂, R³¹ is a protecting group or a reportergroup, R³² and R¹⁷ are independently selected from the group consistingof —H, —(C₁-C₁₀)-alkyl, —(C₂-C₁₀)-alkenyl and —(C₆-C₂₂)-aryl, R¹⁸ isselected from the group consisting of substituted or unsubstituted—(C₁-C₆)-alkyl, unsubstituted —(C₁-C₆)-alkoxy or —(C₁-C₆)-alkoxysubstituted one or more times by a group selected from the groupconsisting of -halogen, p-nitroaryloxy and -cyano, and B is a group offormula I

wherein W is selected independently from X, Y and Z from the groupconsisting of N and CR², Z is selected from the group consisting of Nand C with the proviso that if Z is N, then X independently from W and Yis selected from the group consisting of N and CR³, and Y independentlyfrom W and X is selected from the group consisting of N and CR⁴, and thebond between X and Y is a double bond and the bond between Y and Z is asingle bond, and if Z is C, then X is NR³³, and Y is selected from thegroup consisting of N and CR⁴ and the bond between Z and Y is a doublebond and the bond between X and Y is a single bond, R¹, R², R³ and R⁴are independently selected from the group consisting of —H, —halogen,—OR¹³, —SR¹⁹, —(C₁-C₁₀)-alkyl, —(C₂-C₁₀)-alkenyl, —(C₂-C₁₀)-alkynyl,—NO₂, —NR⁵R⁶, -cyano, and —C(═O)R¹¹, R¹¹ is selected from the groupconsisting of —OH, —(C₁-C₆)-alkoxy, —(C₆-C₂₂)-aryloxy, and NHR¹², R⁵,R⁶, R¹², R¹³, R¹⁹ and R³³ are selected independently from the groupconsisting of —H, —(C₁-C₁₀)-alkyl, —(C₂-C₁₀)-alkenyl, —(C₂-C₁₀)-alkinyl,—(C₆-C₂₂)-aryl, a protecting group and a reporter group, r and s areindependently of each other an integer of 1 to 18, D is the position ofattachment of the group to the rest of the nucleic acid bindingcompound, and alkyl, alkenyl and alkynyl being unsubstituted orsubstituted by one or more moieties selected from the group consistingof -halogen, —S—(C₁-C₆)-alkyl, —(C₁-C₆) -alkoxy, —NR⁵R⁶, —CO—R¹¹,—NH—CO—NR⁵R⁶, —NH—CSNR⁵R⁶ and —[O—(CH₂)_(r)]_(s)—NR⁵R⁶, with the provisothat at least one of R⁵ and R⁶ of —NR⁵R⁶ is a protecting group.
 3. Thecompound of claim 2, wherein R¹ is not OR³¹.
 4. The compound of claim 2,wherein said group of formula I contains at least one reporter group. 5.The compound of claim 2, wherein said group of formula I is selectedfrom the group consisting of groups of formula I, wherein either W is N,Z is N, Y is N and X is CR³, or W is N, Z is C, Y is N and X is CR³, orW is N, Z is N, Y is N and X is N.
 6. A method for the enzymaticsynthesis of the compound of claim 1, comprising reacting a triphosphatesubunit with a primer using a nucleic acid as a template for theelongation of the primer, wherein the triphosphate subunit contains aheterocyclic group of formula I.
 7. The method according to claim 6,wherein said triphosphate subunit has the formula VIII

wherein PPP is a triphosphate group, R¹⁴ is selected from the groupconsisting of —H, —OH, —(C₁-C₁₀)-alkoxy, —(C₂-C₁₀)-alkenyloxy,—(C₂-C₁₀)-alkynyloxy halogen, -azido and NH₂, and B is a group offormula I, with the proviso that R¹⁴ is not OH if B is 2-azaadenine. 8.A compound of the general formula VIII

wherein PPP is a triphosphate group, R¹⁴ is selected from the groupconsisting of —H, —OH, —(C₁-C₁₀)-alkoxy, —(C₂-C₁₀)-alkenyloxy,—(C₂-C₁₀)-alkynyloxy halogen, -azido and NH₂, and B is a group offormula I, with the proviso that R¹⁴ is not OH if B is 2-azaadenine. 9.The compound of claim 8, wherein -M-R¹⁶ is a triphosphate group and-M-R¹⁵ is —OH.
 10. The compound of claim 9, wherein R¹⁴ is —OH.
 11. Thecompound of claim 8, wherein R¹⁴ is —H and R¹ is not OH.
 12. Thecompound of general formula IX

wherein A is selected from the group consisting of O, S andN—(C₁-C₆)-alkyl, M and M′ are independently selected from the groupconsisting of oxy, sulfanediyl, —NR²², —(C₁-C₁₀)-alkyl, or—O—(C₁-C₁₀)-alkyl-O—, and —S—(C₁-C₁₀)-alkyl-O— and—NR²²—(C₁-C₆)-alkyl-O—, R²² is selected from the group of —H and—(C₁-C₁₀)-alkyl, R¹⁴ is selected from the group consisting of —H, —OR³¹,—(C₁-C₁₀)-alkoxy, —(C₂-C₁₀)-alkenyloxy, —(C₂-C₁₀)-alkynyloxy, -halogen,-azido NHR³¹, SR³¹ and —NH₂, R³¹ is a protecting group or a reportergroup, R³² and R¹⁷ are independently selected from the group consistingof —H, —(C₁-C₁₀)-alkyl, —(C₂-C₁₀)-alkenyl and -(C₆-C₂₂)-aryl, R¹⁸ isselected from the group consisting of substituted or unsubstituted—(C₁-C₆)-alkyl, unsubstituted —(C₁-C₆)-alkoxy or —(C₁-C₆)-alkoxysubstituted one or more times by a group selected from the groupconsisting of -halogen, p-nitroaryloxy and -cyano, and B is a group offormula I

wherein W is selected independently from X, Y and Z from the groupconsisting of N and CR², Z is selected from the group consisting of Nand C with the proviso that if Z is N, then X independently from W and Yis selected from the group consisting of N and CR³, and Y independentlyfrom W and X is selected from the group consisting of N and CR⁴, and thebond between X and Y is a double bond and the bond between Y and Z is asingle bond, and if Z is C, then X is NR³³, and Y is selected from thegroup consisting of N and CR⁴ and the bond between Z and Y is a doublebond and the bond between X and Y is a single bond, R¹, R², R³ and R⁴are independently selected from the group consisting of —H, -halogen,—OR¹³, —SR¹⁹, —(C₁-C₁₀)-alkyl, —(C₂-C₁₀)-alkenyl, —(C₂-C₁₀)-alkynyl,—NO₂, —NR⁵R⁶, -cyano, and —C(═O)R¹¹, R¹¹ is selected from the groupconsisting of —OH, —(C₁-C₆)-alkoxy, —(C₆-C₂₂)-aryloxy, and NHR¹², R⁵,R⁶, R¹², R¹³, R¹⁹ and R³³ are selected independently from the groupconsisting of —H, —(C₁-C₁₀)-alkyl, —(C₂-C₁₀)-alkenyl, —(C₂-C₁₀)-alkinyl,—(C₆-C₂₂)-aryl, a protecting group and a reporter group, r and s areindependently of each other an integer of 1 to 18, D is the position ofattachment of the group to the rest of the nucleic acid bindingcompound, and alkyl, alkenyl and alkynyl being unsubstituted orsubstituted by one or more moieties selected from the group consistingof -halogen, —S—(C₁-C₆)-alkyl, —(C₁-C₆)-alkoxy, —NR⁵R⁶, —CO—R¹¹,—NH—CO—NR⁵R⁶, —NH—CSNR⁵R⁶— and —[O—(CH₂)_(r)]_(s)—NR⁵R⁶, with theproviso that at least one of R⁵ and R⁶ of —NR⁵R⁶ is a protecting group.13. A compound of general formula X

wherein M and M′ are independently selected from the group consisting ofoxy, sulfanediyl, —NR²², —(C₁-C₁₀)-alkyl, or —O—(C₁-C₁₀)-alkyl-O—, and—S—(C₁-C₁₀)-alkyl-O— and —NR²²—(C₁-C₆)-alkyl-O—, R²² is selected fromthe group of —H and —(C₁-C₁₀)-alkyl, R¹⁴ is selected from the groupconsisting of —H, —OR³¹, —(C₁-C₁₀)-alkoxy, —(C₂-C₁₀)-alkenyloxy,—(C₂-C₁₀)-alkynyloxy, -halogen, -azido NHR³¹, SR³¹ and —NH₂, R³¹ is aprotecting group or a reporter group, B is a group of formula I

wherein W is selected independently from X, Y and Z from the groupconsisting of N and CR², Z is selected from the group consisting of Nand C with the proviso that if Z is N, then X independently from W and Yis selected from the group consisting of N and CR³, and Y independentlyfrom W and X is selected from the group consisting of N and CR⁴, and thebond between X and Y is a double bond and the bond between Y and Z is asingle bond, and if Z is C, then X is NR³³, and Y is selected from thegroup consisting of N and CR⁴ and the bond between Z and Y is a doublebond and the bond between X and Y is a single bond, R¹, R², R³ and R⁴are independently selected from the group consisting of —H, -halogen,—OR¹³, —SR¹⁹, —(C₁-C₁₀)-alkyl, —(C₂-C₁₀)-alkenyl, —(C₂-C₁₀)-alkynyl,—NO₂, —NR⁵R⁶, -cyano, and —C(═O)R¹¹, R¹¹ is selected from the groupconsisting of —OH, —(C₁-C₆)-alkoxy, —(C₆-C₂₂)-aryloxy, and NHR¹², R⁵,R⁶, R¹², R¹³, R¹⁹ and R³³ are selected independently from the groupconsisting of —H, —(C₁-C₁₀)-alkyl, —(C₂-C₁₀)-alkenyl, —(C₂-C₁₀)-alkinyl,—(C₆-C₂₂)-aryl, a protecting group and a reporter group, r and s areindependently of each other an integer of 1 to 18, D is the position ofattachment of the group to the rest of the nucleic acid bindingcompound, and alkyl, alkenyl and alkynyl being unsubstituted orsubstituted by one or more moieties selected from the group consistingof -halogen, —S—(C₁-C₆)-alkyl, —(C₁-C₆)-alkoxy, —NR⁵R⁶, —CO—R¹¹,—NH—CO—NR⁵R⁶, —NH—CSNR⁵R⁶— and —[O—(CH₂)_(r)]_(s)—NR⁵R⁶.